List of Tables

List of Figures

Glossary

ALS

Amyotrophic lateral sclerosis ALSFRS-R Revised Amyotrophic Lateral Sclerosis Functional Rating scale ASM active surface method CNR CNS imaging, diffusion tensor imaging, magnetization transfer imaging, segmentation, atrophy measurements, surface-based morphometry.

Introduction

The spinal cord is the entry for sensory information toward the Central Nervous System (CNS) and the exit for motor commands from the CNS to the limbs and the trunk. Lesions of the spinal cord due to neurodegenerative diseases thus lead to major sensory and/or motor consequences whose severity depends on the extent of the lesions. Degenerative motor neuron diseases (MND) form a subgroup of neurodegenerative diseases, and are characterized by a progressive dysfunction and loss of ventral horn motoneurons of the spinal grey matter. Beyond this common anatomical susceptibility, which is responsible for a progressive and diffuse weakness, other neurological systems are also impaired. The corticospinal tract (CST) degeneration is a classical feature of amyotrophic lateral sclerosis (ALS), which is the most common adult onset MND, but a more widespread multisystem involvement is now well recognized. In particular, sensory system involvement has been demonstrated in animal models of ALS. Proximal spinal muscular atrophy (SMA) is the second most common MND and shares with ALS a common feature of ventral horn motoneurons loss. The reason of the classical proximal predominance of the deficit in SMA remains unknown.

In human patients, magnetic resonance imaging (MRI) has emerged as the most powerful approach at the brain level to extract quantitative data on neuronal loss, axonal degeneration and demyelination in degenerative conditions. Studies at the spinal cord levels are scarce mainly because of technical and methodological difficulties. Despite, clinical conventional MRI remains extremely useful in describing a large range of spinal cord abnormalities. However, conventional MRI lacks sensitivity and specificity in providing quantitative data on the integrity of white matter tracts and grey matter layers in patients with sensory and/or motor signs detected during clinical and electrophysiological evaluation, which is particularly the case in MND. More advanced and quantitative MRI techniques are used in the research setting, enabling to capture at the spinal level sensory-motor system involvement in MND.

The objective of the present project was to optimize and use advanced multi-parametric MRI approach at the spinal cord level to analyze grey and white matter structures that are impaired in MND, their temporal alterations during the disease course and the functional correlates, as assessed by clinical and electrophysiological examinations.

The manuscript is organized as follows: In Chapter 2, a literature review will present: i) motor neuron diseases with an especial focus on ALS and SMA; ii) spinal cord MRI with an especial focus on advanced MRI techniques that are commonly used in the pathological context. The objectives of this thesis will be presented in Chapter 3. Five published articles, which constitute the core of the present thesis, will be presented in Chapter 4. Finally, Chapter 5 will discuss our findings and future directions.

Background

Motor neuron diseases

Motor neuron diseases

Historical setting

In the second half of the 19th century, with the contribution of other clinicians, Jean-Martin Charcot, a prominent neurologist from Pitié-Salpêtrière Hospital, recognized and described the clinical, anatomical and physiological features of a large spectrum of MND, including ALS, progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS) [START_REF] Goetz | Amyotrophic lateral sclerosis: early contributions of Jean-Martin Charcot[END_REF]. Theses characterizations were based on a rigorous description of 20 cases and five autopsies. J.M. Charcot highlighted the major contribution of lower motor neuron (LMN) loss in muscles weakness and amyotrophy as well as the association with pyramidal tract involvement ("lateral sclerosis") causing spasticity and contractures [START_REF] Goetz | Amyotrophic lateral sclerosis: early contributions of Jean-Martin Charcot[END_REF][START_REF] Katz | Amyotrophic Lateral Sclerosis: A Historical Perspective[END_REF]. Dispite the historical association of ALS recognition with Jean-Martin Charcot, Jacob Augustus Lockhart Clarke, a prominent British physician, described the clinical and the pathophysiological features of an ALS case in a paper published with Charles Bland Radcliffe (Radcliffe and Lockhart Clarke, 1862), 3 years ahead of Charcot's description of this disease (Turner et al., 2010).

In parallel, several familial forms of MND were also described. For instance, by the end of the 19th century, Johan Hoffman and Guido Werdnig described the clinical and the anatomopathological features of new forms of hereditary infantile paralysis, known as infantile SMA (Dubwitz, 2009). Their spinal cord autopsies showed notably ventral roots atrophy and loss of ventral horn motoneurons. Later in the 20's century, Victor Dubwitz, Erik Klas Hendrik Kugelberg, Lisa Welander and Klauss Zerre described the other forms of familial SMA, i.e. intermediate, juvenile and adult SMA (Dubwitz, 1964(Dubwitz, , 1967;;Kugelberg and Welander, 1986;Zerre et al., 1995Zerre et al., , 1997)). DNA sequencing in the 90's allowed associating some familial forms of MND with specific genes mutations, notably in hereditary SMA with survival motor neuron 1 gene deletion (Lefebvre et al., 1995), and in ALS with superoxyde dismutase 1 (SOD1), followed by the identification of an increasing number of causative genes (TAR DNA-binding protein (TDP-43), FUsed in Sarcoma (FUS), chromosome 9 open reading frame 72 (C9ORF72)…) [START_REF] Renton | State of play in amyotrophic lateral sclerosis genetics[END_REF].

In more than 150 years, rigorous and intensive effort was made by several generations of clinicians and researchers to: i) describe the demographical, clinical, pathophysiological and recently the genetic features of the MND forms; ii) figure out if some MND forms are part of a single entity, i.e. progressive bulbar palsy and ALS (Joseph Dejerine in 1883), or could be considered as a continuum spectrum of MND, i.e. PLS, ALS and PMA (Van Den Berg 2003; Swinnen and Robberecht, 2014); iii) develop tools enabling accurate diagnosis of MND as well as predict and monitor disease progression. However, the pathophysiological mechanisms of neurodegeneration are not fully understood in MND, notably in ALS and SMA, which are the two most common forms of human MND observed in adults and in children/young adults, respectively (D'Amico et al., 2011; Wijesekera and Leigh, 2009).

The advent of neuroimaging and particularly magnetic resonance imaging (MRI) by the end of 80's provided new insights into the pathophysiology of MND [START_REF] Goodin | Magnetic resonance imaging in amyotrophic lateral sclerosis[END_REF]. MRI allows a noninvasive and in-vivo investigation of the structural (structural imaging) and functional (functional imaging) integrity of the CNS during the course of MND [START_REF] Unrath | Whole brain-based analysis of regional white matter tract alterations in rare motor neuron diseases by diffusion tensor imaging[END_REF][START_REF] Shen | A Systematic Review and Meta-Analysis of the Functional MRI Investigation of Motor Neuron Disease[END_REF][START_REF] Turner | What does imaging reveal about the pathology of amyotrophic lateral sclerosis?[END_REF]. Notably, MRI enables investigation of the motor system structures that are classically impaired in MND [START_REF] Rocha | Is magnetic resonance imaging a plausible biomarker for upper motor neuron degeneration in amyotrophic lateral sclerosis/primary lateral sclerosis or merely a useful paraclinical tool to exclude mimic syndromes? A critical review of imaging applicability in clinical routine[END_REF]. It also allows assessing the anatomo-functional degenerative process beyond the motor system (van der Graaff et al., 2008;Chiò et al., 2012).

Particularly, many studies used MRI to investigate the anatomic substrate of cognitive symptoms in ALS [START_REF] Rajagopalan | Distinct patterns of cortical atrophy in ALS patients with or without dementia: an MRI VBM study[END_REF]Schuster et al., 2014;[START_REF] Ambikairajah | A visual MRI atrophy rating scale for the amyotrophic lateral sclerosis-frontotemporal dementia continuum[END_REF].

Amyotrophic lateral sclerosis

Anatomopathology Amyotrophic lateral sclerosis is the most common MND. ALS is characterized by a progressive loss of upper motor neurons (UMN), located at primary motor cortex, i.e. pyramidal cells, and LMN, which are the brainstem motor nuclei and alpha motoneurons located in the anterior horn of the spinal cord (Figure 1).

At the spinal cord level, in addition to the lateral CST degeneration, the anterior/ventral CST is also involved [START_REF] Swash | Selective and asymmetric vulnerability of corticospinal and spinocerebellar tracts in motor neuron disease[END_REF]. At the brain level, animal and human studies show evidence of involvement of other structures beyond primary motor cortex, such as the prefrontal cortex, the supplemental motor area, the primary and secondary somatosensory cortex, the basal ganglia and the cerebellum. All these findings support the concept of ALS as a multi-system neurodegenerative disease ( [START_REF] Bede | The selective anatomical vulnerability of ALS: 'disease-defining' and 'disease-defying' brain regions[END_REF].

Incidence and prevalence

The worldwide annual incidence of ALS is 1.75 (range = 1.55-1.96)/100,000 individuals, 2.03 (1.79-2.37) in men and 1.45 (1.25-1.64) in women [START_REF] Marin | Variation in worldwide incidence of amyotrophic lateral sclerosis: a meta-analysis[END_REF]) (Figure 2). In Europe, crude incidence ranges from 1.92 (1.49-2.34) in northern Europe to 2.35 (1.79-2.92) in western Europe per 100,000 individuals [START_REF] Marin | Variation in worldwide incidence of amyotrophic lateral sclerosis: a meta-analysis[END_REF]. ALS incidence increases with age, with a peak between 60 and 75 years of age [START_REF] Marin | Variation in worldwide incidence of amyotrophic lateral sclerosis: a meta-analysis[END_REF][START_REF] Chiò | Global epidemiology of amyotrophic lateral sclerosis: a systematic review of the published literature[END_REF].

The peak of incidence for men is at 70-74 years and at 65-69 years for women (Logroscino et al., 2015). The gender ratio of incidence between men and women is 1.3:1 (Logroscino et al., 2015).

The prevalence of ALS is subject to large variations among studies, ranging from 1.1/100,000 population in Yugoslavia [START_REF] Alcaz | Epidemiological and clinical characteristics of ALS in Belgrade, Yugoslavia[END_REF] to 8.2/100,000 in Faroe Islands [START_REF] Joensen | Incidence of amyotrophic lateral sclerosis in the Faroe Islands[END_REF].

Genetic features

The role of genetic factors in ALS has been demonstrated by the identification of the SOD1 gene mutation in 1993 [START_REF] Rosen | Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis[END_REF]. Since then, more than twenty gene mutations have been found in familial ALS [START_REF] Yamashita | Genotype-phenotype relationship in hereditary amyotrophic lateral sclerosis[END_REF], the most important being C9ORF72 which was found in nearly 40% of familial cases and about 6% of sporadic forms. Familial ALS represents about 10% of ALS cases (Bayrn et al., 2011). The inheritance is mostly autosomal dominant with rare autosomal recessive and recessive X-linked modes [START_REF] Andersen | Clinical genetics of amyotrophic lateral sclerosis: what do we really know?[END_REF].

Familial ALS forms are characterized by an earlier age at onset compared to sporadic forms (49.7 ± 12.3 versus 61.8 ± 3.8 years), with a median survival of 2.7 years, a gender ratio of 1 to 1.3:1, and a predominance of lower limbs onset [START_REF] Bali | Defining SOD1 ALS natural history to guide therapeutic clinical trial design[END_REF][START_REF] Camu | Genetics of familial ALS and consequences for diagnosis. French ALS Research Group[END_REF]. Familial ALS disease duration has a bimodal profile: a small number of patients have a very poor prognosis (< 2 years), and most have better prognosis than the sporadic form (> 5 years) [START_REF] Camu | Genetics of familial ALS and consequences for diagnosis. French ALS Research Group[END_REF].

Pathophysiology

Multiple pathogenic mechanisms implicated in neurodegeneration in ALS were identified. The most likely neurodegeneration mechanisms are: protein aggregation, neuronal inflammation, oxidative stress, and motoneurons hyper-excitability induced by glutamate excess. In parallel, several mechanisms were proposed to explain the spreading of ALS symptoms.

Protein aggregate

ALS shares the presence of protein aggregates that form inclusions in motoneurons with many neurodegenerative diseases [START_REF] Ross | Protein aggregation and neurodegenerative disease[END_REF][START_REF] Kumar | Protein aggregation and neurodegenerative diseases: From theory to therapy[END_REF]. The TDP-43 is the main component of these protein aggregates, which is also mutated in some familial forms of ALS [START_REF] Sreedharan | TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF]. The TDP-43 accumulates in neurons and non-neuronal cells in sporadic forms and the familial forms, except for familial ALS with SOD1 mutation [START_REF] Mackenzie | Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations[END_REF]. Other proteins form these aggregates: cystatin C and transferrin in Bunina bodies, SOD1 or protein FUS/Translated in liposarcoma (FUS/TLS). These aggregates are secondary to impaired protein degradation components (proteasome or autophagy mechanisms).

Altered ARN metabolism

Altered ARN mechanism was suggested in ALS because TDP-43 and FUS, which are mutated in ALS, are involved in ARN splicing, transport and translation [START_REF] Verma | Altered RNA metabolism and amyotrophic lateral sclerosis[END_REF]. Focal accumulations of RNA in nuclei exist in the C9ORF72 gene mutation, which block transcription factors and disrupt cell viability [START_REF] Ishiura | Epidemiology and molecular mechanism of frontotemporal lobar degeneration/amyotrophic lateral sclerosis with repeat expansion mutation in C9orf72[END_REF].

Neurotransmitters and motoneurons afferent abnormalities

Neuronal excitotoxicity was the topic of significant research, despite the fact this mechanism is considered as a delayed mechanism in neurodegeneration [START_REF] King | Excitotoxicity in ALS: Overstimulation, or overreaction?[END_REF]. The main arguments supporting neuronal excitotoxicity are: increased glutamate level in the cerebrospinal fluid (CSF) [START_REF] Spreux-Varoquaux | Glutamate levels in cerebrospinal fluid in amyotrophic lateral sclerosis: a reappraisal using a new HPLC method with coulometric detection in a large cohort of patients[END_REF], and the effect of riluzole [START_REF] Lacomblez | A confirmatory dose-ranging study of riluzole in ALS. ALS/Riluzole Study Group-II[END_REF]. Neuronal death was linked to calcium accumulation in neuronal cells due to a relative defect of calcium degradation systems.

Recently a great attention has been given to motoneuron afferent and neurotransmitter disturbances in ALS [START_REF] Pradat | Neuroprotective strategies in amyotrophic lateral sclerosis: modulation of neurotransmittory and neurotrophic input to motoneurons[END_REF]. Several afferents project directly or through interneurons into LMN and UMN, which determines their excitability condition, function and survival.

Dependent GABA inhibitory cortical pathway abnormalities have been demonstrated in Positron

Emission Tomography (PET) [START_REF] Lloyd | Extramotor involvement in ALS: PET studies with the GABA(A) ligand [(11)C]flumazenil[END_REF] and NMR spectroscopy (Foerster et al., 2012).

Abnormal spinal glycinergic inhibitory systems could lead to an increase in motoneurons excitability [START_REF] Chang | Glycine receptor channels in spinal motoneurons are abnormal in a transgenic mouse model of amyotrophic lateral sclerosis[END_REF]. Human and animal model studies suggested that impaired afferents from neuromuscular spindles contribute to excitability aggravation [START_REF] Malaspina | Activation transcription factor-3 activation and the development of spinal cord degeneration in a rat model of amyotrophic lateral sclerosis[END_REF]Iglesis et al., 2015).

Implication of non-neuronal cells and neural inflammation

Animal models showed that neurodegeneration in ALS is not a cell-autonomous process [START_REF] Clement | Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice[END_REF] but depends on astrocytes, microglia, oligodendrocytes and Schwann cells. It is important to mention that inflammatory reaction related to microglia can be toxic but also neuroprotective [START_REF] Beers | Wildtype microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis[END_REF][START_REF] Xiao | Mutant SOD1(G93A) microglia are more neurotoxic relative to wild-type microglia[END_REF]. Moreover, several studies highlighted the possible implication of the neuromuscular junction and muscle in the pathogenesis of motoneuron degeneration [START_REF] Dupuis | Nogo provides a molecular marker for diagnosis of amyotrophic lateral sclerosis[END_REF][START_REF] Fischer | Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man[END_REF][START_REF] Bruneteau | Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in reinnervation ability and disease progression[END_REF][START_REF] Pradat | Muscle Nogo-A expression is a prognostic marker in lower motor neuron syndromes[END_REF][START_REF] Pradat | Abnormalities of satellite cells function in amyotrophic lateral sclerosis[END_REF].

Spread mechanisms

It has been recently suggested that the regional spread of neurodegeneration during the disease progression is associated with prion-like (proteinaceous infectious particles) propagation mechanisms [START_REF] Pradat | Deciphering spreading mechanisms in amyotrophic lateral sclerosis: clinical evidence and potential molecular processes[END_REF], Juker et al., Nature 2013). In ALS, prion-like mechanisms could explain the intercellular spread by neuroanatomical networks, the trans-synaptic passing of mutated proteins or by the spreading of toxic factors throughout the cerebrospinal fluid or blood [START_REF] Pradat | Deciphering spreading mechanisms in amyotrophic lateral sclerosis: clinical evidence and potential molecular processes[END_REF].

Additional mechanisms

Additional mechanisms are involved in motoneurons loss: oxidative stress, mitochondrial dysfunction, disruption of axonal transport and endoplasmic reticulum stress [START_REF] Robberecht | The changing scene of amyotrophic lateral sclerosis[END_REF].

Phenotypes

Amyotrophic lateral sclerosis is a heterogeneous condition with eight recognized phenotypes [START_REF] Ludolph | A revision of the El Escorial criteria -2015[END_REF][START_REF] Chiò | PARALS study group. Phenotypic heterogeneity of amyotrophic lateral sclerosis: a population based study[END_REF], according to the site of degeneration (bulbar, cervical or lumbar) and the balance between UMN and LMN degeneration (Figure 3): 1) classical phenotype (as described by Charcot); 2) bulbar phenotype; 3) flail arm phenotype; 4) flail leg phenotype; 5) respiratory phenotype; 6) predominant UMN phenotype; 7) pure LNM phenotype; 8) pure UMN phenotype. Principal characteristics of the eight phenotypes are summarized in Table 1.

Classical (Charcot's) phenotype

Charcot initially described the classical ALS form. The mean age at onset is 63 years, with a gender ratio of incidence between men and women of 1.65:1. This form is characterized by progressive amyotrophic motor weakness in the upper limbs, often asymmetrical, associated with a pyramidal syndrome without important lower limbs atrophy. Bulbar involvement occurs later in the disease course, and is associated with an emotional lability and sometimes with a pseudo-bulbar syndrome.

Median of survival from onset is 2.6 years with 10% survival rate after 10 years from the symptoms onset [START_REF] Chiò | PARALS study group. Phenotypic heterogeneity of amyotrophic lateral sclerosis: a population based study[END_REF].

Flail arm

Flail arm phenotype is characterized by upper limbs deficit due to motoneurons degeneration. This phenotype is associated with delayed moderate signs of UMN involvement. The mean age at onset is 63 years, with a gender ratio of incidence between men and women of 4:1. Disease progression is slow, with a median of survival from the onset of 4.0 years and a survival rate of 17.4% at 10 years [START_REF] Chiò | PARALS study group. Phenotypic heterogeneity of amyotrophic lateral sclerosis: a population based study[END_REF].

Flail leg phenotype or Marie-Patrikios pseudo-polyneuritic form

Flail leg phenotype is characterized by unilateral and distal weakness and wasting in the lower limbs. Involvement starts at the distal part of the leg and progresses to the proximal one. The contralateral leg is involved after few weeks or months from the onset of symptoms. Disease progression is slow, with a median survival from the onset of 3.0 years and a survival rate of 12.8% at 10 years, which is similar to the Charcot's form [START_REF] Chiò | PARALS study group. Phenotypic heterogeneity of amyotrophic lateral sclerosis: a population based study[END_REF].

Bulbar phenotype

Disease onset is characterized by a dysphagia or dysarthria, rapidly associated with pseudo-bulbar signs with yawning, spasmodic laughter and cries. The mean age at onset is 69 years, higher than in the classical syndrome, without gender incidence predominance in men. Median of survival from the onset is 2.0 years, shorter than the classical syndrome, with only 3.4% survival rate at 10 years from the symptoms onset [START_REF] Chiò | PARALS study group. Phenotypic heterogeneity of amyotrophic lateral sclerosis: a population based study[END_REF].

Respiratory phenotype

Respiratory impairment is rarely the predominant symptom at onset in ALS [START_REF] Mitsumoto | Clinical features: signs and symptoms[END_REF]. However, in some cases respiratory impairment can be the revealing symptom. The mean age at onset in these respiratory forms is 62 years, with predominance in men (gender ratio of incidence 6:1).

Respiratory insufficiency is revealed by signs of chronic alveolar hyperventilation [START_REF] Howard | Respiratory complications and their management in motor neuron disease[END_REF] or by a severe acute respiratory failure that requires rapid initiation of artificial ventilation ( [START_REF] De Carvalho | Motor neuron disease presenting with respiratory failure[END_REF]. Neurological examination frequently reveals other signs of motoneurons involvement [START_REF] Howard | Respiratory complications and their management in motor neuron disease[END_REF], although they can be absent in some cases. This phenotype is highly progressive, with a median of survival of 1.4 years and no patients survive after 10 years from the onset [START_REF] Chiò | PARALS study group. Phenotypic heterogeneity of amyotrophic lateral sclerosis: a population based study[END_REF].

Predominant UMN phenotype or pyramidal phenotype

Pyramidal signs are predominant and are associated with a moderate motor deficit and a marked hypertonia in the lower limbs. Patients show slow progression compared to those with the classical phenotype, with a median of survival of 6.3 years and 31.9% survival rate at 10 years from the onset [START_REF] Chiò | PARALS study group. Phenotypic heterogeneity of amyotrophic lateral sclerosis: a population based study[END_REF].

Pure LMN phenotype or PMA

The phenotype is clinically characterized by diffuse and exclusive LMN degeneration. However, in a study on 91 PMA patients [START_REF] Kim | Study of 962 patients indicates progressive muscular atrophy is a form of ALS[END_REF], 20 patients developed UMN signs after a median follow-up of 23.4 months from diagnosis. Post-mortem studies show additional evidence of the CST and UMN degeneration in PMA [START_REF] Leung | Diagnosis of ALS: clinicopathologic analysis of 76 au-topsies[END_REF][START_REF] Ince | Corticospinal tract degeneration in the progressive muscular atrophy variant of ALS[END_REF], which lead to the inclusion of PMA in the ALS spectrum. Progression in PMA is slow, with a median survival of 7.3 years [START_REF] Chiò | PARALS study group. Phenotypic heterogeneity of amyotrophic lateral sclerosis: a population based study[END_REF]. Gender ratio of incidence is higher in men (2.6:1) [START_REF] Kim | Study of 962 patients indicates progressive muscular atrophy is a form of ALS[END_REF].

Pure UMN phenotype or PLS

Upper motor neurons degeneration clinical signs are exclusive in this phenotype: pyramidal syndrome predominating in lower limbs and pseudo-bulbar syndrome, without significant muscular atrophy. Patients show slow disease progression, and at least 4 years delay is needed to observe all previous clinical signs, and generally LMN degenerating occurs before this delay [START_REF] Gordon | The natural history of primary lateral sclerosis[END_REF].

Post-mortem examination showed evidence of LMN in most cases, which suggest that PLS is part of the ALS spectrum (D' [START_REF] D'amico | Clinical evolution of pure upper motor neuron disease/dysfunction (PUMMD)[END_REF]. Among all ALS phenotypes, PLS has the longest disease evolution, with median survival of 13 years and 71% survival rate at 10 years from symptom onset [START_REF] Chiò | PARALS study group. Phenotypic heterogeneity of amyotrophic lateral sclerosis: a population based study[END_REF].

Epidemiology

Amyotrophic lateral sclerosis is classically described as late-onset disease, with a mean ± SD age at onset of 61.8 ± 3.8 years (range 54-67) [START_REF] Chiò | Global epidemiology of amyotrophic lateral sclerosis: a systematic review of the published literature[END_REF]. Mean age at onset for men is 60.8 years and 61.9 years for women (McCombe and Henderson, 2010). Median survival from onset ranges from 20 to 48 months [START_REF] Marin | Variation in worldwide incidence of amyotrophic lateral sclerosis: a meta-analysis[END_REF][START_REF] Chiò | Aosta Register for ALS (PARALS). Early symptom progression rate is related to ALS outcome: a prospective population-based study[END_REF][START_REF] Chiò | Global epidemiology of amyotrophic lateral sclerosis: a systematic review of the published literature[END_REF]Gordon 2013). However, in 10 to 26.6% cases survival rate exceeds 5 years and, in 5 to 13.3%, 10 years from symptom onset [START_REF] Chiò | Epidemiology of ALS in Italy: a 10-year prospective population-based study[END_REF][START_REF] Pupillo | Long-term survival in amyotrophic lateral sclerosis: a population-based study[END_REF].

Age is a strong prognostic factor in ALS, with a survival time inversely correlated with age at onset [START_REF] Chiò | Epidemiology of ALS in Italy: a 10-year prospective population-based study[END_REF]. For instance, patients with onset before the age of 40 years have longer median survival, often more than 10 years [START_REF] Testa | Survival of 793 patients with amyotrophic lateral sclerosis diagnosed over a 28-year period[END_REF]del Aguila 2003), and after 80 years, median survival is substantially reduced to less than 2 years [START_REF] Forbes | Scottish ALS/MND Register. The epidemiology of amyotrophic lateral sclerosis (ALS/MND) in people aged 80 or over[END_REF].

Clinical evolution

Progressive disability worsening is an important criterion for ALS diagnosis. The median survival from symptom onset ranges from 20 to 48 months [START_REF] Marin | Variation in worldwide incidence of amyotrophic lateral sclerosis: a meta-analysis[END_REF][START_REF] Chiò | Aosta Register for ALS (PARALS). Early symptom progression rate is related to ALS outcome: a prospective population-based study[END_REF][START_REF] Chiò | Global epidemiology of amyotrophic lateral sclerosis: a systematic review of the published literature[END_REF]Gordon 2013). Disease duration in ALS is heterogeneous and mainly depends on the phenotype and age at onset; some ALS patients will have a very fast disease progression and others a very long and slow one, with 10 to 26.6% of cases surviving more than 5 years from onset [START_REF] Chiò | Epidemiology of ALS in Italy: a 10-year prospective population-based study[END_REF][START_REF] Pupillo | Long-term survival in amyotrophic lateral sclerosis: a population-based study[END_REF].

Consequently, ALS diagnosis should systematically be considered even with the absence of worsening after a 12 months from the onset.

The most common causes of death in ALS patients are a respiratory failure (77 %), which is usually linked to a terminal respiratory insufficiency (58%), and pneumonia favored by deglutition disorders (14 %) [START_REF] Gil | Causes of death amongst French patients with amyotrophic lateral sclerosis: a prospective study[END_REF]. However, causes of death are not known in 13% of cases [START_REF] Gil | Causes of death amongst French patients with amyotrophic lateral sclerosis: a prospective study[END_REF].

Rulizole is the only treatment that has proved effective to slow ALS disease progression [START_REF] Lacomblez | A confirmatory dose-ranging study of riluzole in ALS. ALS/Riluzole Study Group-II[END_REF]. Treatment action mechanisms are poorly understood and are probably numerous, involving at least altered glutamate metabolism and sodium channel function modulation [START_REF] Cheah | Riluzole, neuroprotection and amyotrophic lateral sclerosis[END_REF][START_REF] Vucic | Riluzole exerts central and peripheral modulating effects in amyotrophic lateral sclerosis[END_REF]. Taken orally twice a day, a 100 mg/day dose (50 mg morning and evening), riluzole decreases the relative risk of death or tracheostomy by 35% over a period of 18 months after adjustment for risk factors [START_REF] Lacomblez | A confirmatory dose-ranging study of riluzole in ALS. ALS/Riluzole Study Group-II[END_REF].

Para-clinical examinations

Electromyography and sensory evoked potentials

Electrophysiology examination is the key tool for the positive and differential diagnosis of ALS.

Electromyography produces quantitative measures of LMN degeneration, confirming detected signs at the clinical examination at an early stage even in patients with predominant or pure UMN phenotype (de [START_REF] Carvalho | Electrodiagnostic criteria for diagnosis of ALS[END_REF]. Needle examination includes the detection of abnormal activity at rest reflecting denervation such as fibrillation and fasciculation potentials. According to the new diagnosis criteria of Awaji-Shima, involvement signs in different anatomical regions detected by electromyography (bulbar, cervical, thoracic and lumbar) have the same diagnosis value as those from clinical examination (de [START_REF] Carvalho | Electrodiagnostic criteria for diagnosis of ALS[END_REF]. Conduction velocity is normal and conduction blocks are not detected, excluding a classic differential diagnostic of multifocal motor neuropathy with conduction blocks. The decrease of sensory evoked potentials (SEP), especially in the lower limbs, lead to suspect a spinal and bulbar muscular atrophy and to carry out a genetic testing [START_REF] Pradat | Atrophie bulbo-spinale liée à l'X : une maladie rare mais un diagnostic différentiel fréquent de la sclérose latérale amyotrophique[END_REF]. Sensory evoked potentials are useful in the differential diagnosis measure but the detection of abnormalities does not exclude the diagnosis of an ALS (Iglesias et al., 2015)

Motor evoked potential

Transcranial magnetic stimulation (TMS) is a noninvasive brain stimulation technique. This technique generates a changing magnetic field pulses that penetrates the scalp and the skull to reach the small cortex area below the coil center. These pulses induce a secondary ionic current flow that activates neurons at the stimulated site, e.g. primary motor cortex in ALS, and evoking indirect waves throughout their axons up to the muscle [START_REF] Hallett | Transcranial Magnetic Stimulation: A Primer[END_REF]. TMS is used to investigate the excitability of brain, brainstem and spinal cord regions, i.e. UMN and LMN. Resulting measures from TMS are motor evoked potential (MEP) conduction time, amplitude and threshold, recruitment curve, cortical silent period and intracortical inhibition and facilitation [START_REF] Hallett | Transcranial Magnetic Stimulation: A Primer[END_REF].

Transcranial magnetic stimulation is used to reveal UMN degeneration at an early stage of ALS even in predominant LMN form, which is clinically difficult to detect. However, the sensitivity of TMS, especially at an early stage of the disease, is debated and this technique is highly dependent on the experience of the examiner, limiting its generalization as a diagnostic tool in clinical practice (de [START_REF] Carvalho | Electrodiagnostic criteria for diagnosis of ALS[END_REF]. Triple stimulation technique is a variant of TMS, and was developed to reduce the variability of MEP amplitude and to increase the sensitivity of MEP measures compared to conventional methods (de [START_REF] Carvalho | Electrodiagnostic criteria for diagnosis of ALS[END_REF].

MRI

Conventional brain and spinal cord MRI is mainly used to exclude "ALS-mimic" syndromes such as cervical spondylotic myelopathy, which is a very common syndrome in seniors (narrow spinal canal associated with a myelopathy intramedullary T2-hypersignal) or, more rarely, a Hirayama disease ("snake eyes" T2*-hypersignal appearance on the cervical anterior horns) ( In two recent papers, we performed a systematic review of MRI findings in ALS from conventional and advanced imaging, and their clinical correlations, and sought to establish whether particular techniques may have value as surrogate biomarkers in this disease as well as their potential use in clinical routine: -Inside the CNS: interneurons, sensory neurons, astrocytes and microglia.

Spinal muscular atrophy

-Outside the CNS: Schwann cells, neuromuscular junction and muscle cells, muscle specific macrophage cells, heart, liver, vascular system, bones, pancreas, lung and intestine.

Previous findings support the concept of SMA, as for ALS (see previous section), as a multisystem disorder (Figure 5).

Genetics of SMA

SMA is caused in 95% of cases by homozygous deletions or mutations of exon 2).

Indeed, the SMN2 gene copy number is inversely correlated with disease severity [START_REF] Swoboda | Natural history of denervation in SMA: relation to age, SMN2 copy number, and function[END_REF]. However, the molecular and cellular mechanisms linking SMN1 gene mutation with the "preferential" LMN loss in SMA remains unclear [START_REF] Errico | Selective vulnerability of spinal and cortical motor neuron subpopulations in delta7 SMA mice[END_REF].

Clinical features and evolution

SMA is classified into four types based on the age at onset and the maximum achieved gross motor function (Zerres et al., 1995[START_REF] Zerres | A collaborative study on the natural history of childhood and juvenile onset proximal spinal muscular atrophy (type II and III SMA): 569 patients[END_REF]. The spectrum of SMA ranges from the most severe (type I), in which patients never manage the ability to sit unassisted and the majority died before two years, to lateonset (types IIIb and IV), in which patients have slow disease progression and normal life expectancy (Zerres et al., 1995;Piepers et al., 2008). In all SMA types, cognition remains intact during the course of the disease.

SMA types

The International SMA Consortium meeting (26-28 June 1992, Bonn, Germany), defined three homogeneous groups of SMA disease (types), based on age at onset of symptoms, disease severity and progression criteria [START_REF] Munsat | International SMA consortium meeting[END_REF]. This classification was further enriched by two population-based studies on SMA (Zerres et al., 1995[START_REF] Zerres | A collaborative study on the natural history of childhood and juvenile onset proximal spinal muscular atrophy (type II and III SMA): 569 patients[END_REF]. Four SMA types are actually recognized:

SMA type I

SMA type I, infantile-onset Werdnig-Hoffman disease, is the most common genetic cause of infantile death. SMA type I is the most severe and common among SMA types (60% of total SMA). The annual incidence for SMA type I ranges from 3.5 to 7.1/100,000 live births with a prevalence ranging from 0.1 to 0.15/100,000 total population [START_REF] Jones | Systematic review of incidence and prevalence of spinal muscular atrophy (SMA)[END_REF]. SMA type I starts before the achievement of walking, with an onset before 6 months. Type I is clinically characterized by profound hypotonia and a severe motor deficit extended to the four limbs and trunk. Patients are not able to sit unassisted, develop tongue-swallowing, weakness, and usually die from respiratory failure within the first 2 years (32% and 8% of survival probability at 2 and 10 years, respectively).

SMA type II

SMA type

SMA type IV

SMA type IV refers to adult-onset (>18 years) and represents less than 5% of total SMA patients.

Patients are clinically characterized by mild proximal muscle weakness and wasting. SMA type IV symptoms are similar to those of type IIIb but with less motor weakness severity. Patients with SMA type IV have normal life expectancy.

Incidence and prevalence

The worldwide annual incidence for all types of SMA ranges from 5.1 to 16.6 cases per 100,000 live births. Annual incidence for SMA types I, II and III, ranges from 3.5 to 7.1, 1.0 to 5.3 and 1.5 to 4.6 cases per 100,000 live births, respectively [START_REF] Jones | Systematic review of incidence and prevalence of spinal muscular atrophy (SMA)[END_REF].

Prevalence per 100,000 total population for SMA types I, II and III, ranges from 0.1 to 0.15, 0.57 to 0.67, and 0.64 to 1.05, respectively [START_REF] Jones | Systematic review of incidence and prevalence of spinal muscular atrophy (SMA)[END_REF]. 6).

Para-clinical examinations

Spinal cord MRI

Principales of MRI

Uniform magnetic field within the scanner is continuously applied, aligning the net magnetic moment M of the tissues protons, mainly hydrogen atoms, which are most abundant protons in the body. Brief radiofrequency pulses of a variable forms tilts M, leading to their free induction decay, and generates current within the received coil around tissues of interest. The decay between the excitation and the return to equilibrium of M is exploited to generate contrast difference between tissues. MR signal is detected using dedicated coils. The protons' density, T1 (spin-lattice), T2 (spin-spin) and T2* (local magnetic field inhomogeneities) relaxations vary from one tissue to another, giving rise to various contrasts and sequences, i.e. T1-, T2-, T2*-weighted sequences) (Table 3). By using different MR parameters, e.g. echo time (TE) and repetition time (TR), different weightings can be obtained, hence revealing contrast deference between tissues. T1-and T2-weighted sequences designate different measurements of protons' relaxations after the application of radiofrequency pulses. The T1-weighted sequence gives arise to a high contrast difference between white and grey matter. T2-weighted sequence is more sensitive to tissues' water content, thus it generates an excellent contrast difference between CSF and white matter. T2*-weighted sequences, derived from T2 decay, considered as "observed" or, "effective" T2 decay, and resulting principally from magnetic field inhomogeneities. T2*-weighted imaging generates high contrast difference between CSF, white and grey matter.

Despite its expensive price, MRI has several advantages over other imaging modalities such as Xray, CT, SPECT and PET: (i) noninvasive nature; (ii) multiplanar imaging possibilities; (iii) high soft tissues conspicuity; (iv) ability to picture at macroscopic scale neuronal content integrity. Over the years, MRI has established itself as the modality of choice to evaluate the CNS integrity in a large range of pathologies, particularly at the brain level. However, imaging the spinal cord is more challenging than that of the brain because of (i) the small cross sectional area of the cord (1 cm diameter) requiring higher spatial resolution and thus decreasing the signal-to-noise ratio (SNR) (see foot-note on next page), (ii) motion from the cerebrospinal fluid flow with each cardiac and respiratory cycle, (iii) partial volume effects at the interface between white/grey matter and white matter/CSF, (iv) susceptibility to artifacts from surrounding tissues (Stroman et al., 2014).

Challenges

Adult human spinal cord has the geometry of a bent elliptic cylinder with 0.8-1.4 cm transverse diameter, 0.5-0.9 cm anterior-posterior diameter, and 43-45 cm length (Fradet et al., 2013;[START_REF] Goto | Development and anatomy of the spinal cord[END_REF]. The spinal cord is surrounded by the CSF, vertebrae and cartilaginous disks, and is close to visceral organs. The major imaging difficulties come from the fact that the spinal cord has tiny crosssectional dimensions in the axial plane, is long in the sagittal/coronal planes, is surrounded by tissues that have different magnetic susceptibilities, and has nearby organs that are prone to motion (Figure 7). In this regard, the spinal cord is considered as one of the most challenging environments for using MR in the human body.

Several studies (Cohen-Adad et al., 2007[START_REF] Verma | Altered RNA metabolism and amyotrophic lateral sclerosis[END_REF]Stroman et al., 2014) defined the major challenges for spinal cord imaging as: i) the partial volume effect; ii) the inhomogeneous magnetic field environment; iii) the physiological and patient motion.

Partial volume effect

The partial volume appears when several tissues are contributing to the same voxel. For the spinal cord imaging it is the case when a voxel is at the interface CSF/white matter, white/grey matter, and eventually CSF/white matter/blood vessels. The MRI signal is then the sum of all signals originated by tissues with different amounts of spins, which leads to a blurry image at boundaries. However, partial volume can be limited by reducing volume sampling, i.e. increasing spatial resolution 1 , but this can result in lower SNR 2 and contrast-to-noise 3 ratios (CNR). An increase of MRI magnet strength (1.5T, 3T or 7T) and/or the number of phased-array coil if parallel imaging is used to decrease the TE and to 1 Spatial resolution: The smallest volume unit detectable by an MRI (voxel). Voxel size is defined by the acquisition field-ofview size, divided by the acquisition matrix size. 2 Signal-to-noise ratio: The ratio between the amplitude of the received signal, divided by the background noise signal that contaminates the image. 3 Contrast-to noise ratio: The signal intensity difference between different tissues in the image, scaled to image noise. the periodical movement can be avoided but this technique implies a longer acquisition time (Taso et al., 2014, Cohen-Adad et al., 2011). Motion artifacts can also be reduced using "saturation bands" that cover the chest and abdomen, by attenuating signals from moving structures so that it does not corrupt the signal from the spinal cord (Figure 9). Velocity compensating gradients sequences and signal averaging across multiple phases of motion can also be applied to minimize motion artifacts. Reducing acquisition time using fast sequences, i.e. fast-spin-echo, paralleled imaging that increases acquisition speed by factors of 1.5 to 3, i.e. SENSE/GRAPPA-type reconstructions, and partial Fourier imaging enable reducing physiological as well as subject motion [START_REF] Jaermann | SENSE-DTI at 3 T[END_REF][START_REF] Glockner | Parallel MR imaging: a user's guide[END_REF] Coregistration of all data when dealing with multiple series acquisition, e.g. diffusion tensor imaging and functional MRI, could also be performed to limit inconsistency in derived maps [START_REF] Mohammadi | The impact of post-processing on spinal cord diffusion tensor imaging[END_REF][START_REF] Vahdat | Simultaneous Brain-Cervical Cord fMRI Reveals Intrinsic Spinal Cord Plasticity during Motor Sequence Learning[END_REF].

Inhomogeneous magnetic field environment

The spinal cord is surrounded by bones and cartilaginous disks. It is also close to the lungs, each containing air. These three elements each have a different magnetic susceptibility. The magnetic field in the spinal cord region is therefore inhomogeneous, leading to geometric distortions and signal intensity loss. These artifacts can be counteracted with shimming. Shimming consists of compensating for the main field inhomogeneities by creating an auxiliary magnetic field via shim coils [START_REF]Electrical current shims for correcting magnetic fields[END_REF]Romeo and Hoult, 1984). However, while volume shimming improves the field homogeneity, it is limited to smooth variations across space and cannot fully compensate for small and localized field variations, such as at cartilaginous discs between vertebral bodies. Echo planar imaging sequence, i.e. diffusion tensor imaging (DTI) (Figure 10), is particularly sensitive to geometric distortions in regions around vertebral disks. In addition to shimming, parallel imaging as well as slices positioning should be considered to limit magnetic field inhomogeneity, i.e. slices centered in the middle of each vertebral body and perpendicular to the spinal cord (Cohen-Adad et al., 2011). The geometry of the magnetic field inhomogeneity should be considered to correct its effect [START_REF] Zeng | Image distortion correction in EPI: comparison of field mapping with point spread function mapping[END_REF][START_REF] Samson | Tissue-and column-specific measurements from multi-parameter mapping of the human cervical spinal cord at 3 T[END_REF].

Clinical setting

Clinical neuroradiology is based on the visual inspection of the CNS MR images, to search for abnormalities that might explain signs detected on clinical and electrophysiological evaluation (Table 4).

Conventional clinical MRI spinal cord protocols are usually composed by fast MRI sequences; two 2D sagittal T1-and T2-weighted sequences with large FOV and low in-plan resolution (typically 1-2 mm2 and 4-5 mm slice thickness) to visually identify and localize involved spinal cord regions, associated with a 2D T2*-weighted sequence with limited FOV and high in-plan resolution (typically 0.5"0.5 mm and 4-5 mm slice thickness) to visually identify the involved spinal cord compartments (white matter, grey matter or nerve roots). Sequences' parameters adjustments are usually limited to FOV positioning without a focal shimming and no saturation bands or gating are used, which limit spinal cord 4).

Syringomyelia

Thin light T2-Hypersignal inside the spinal cord corresponding to CSF obstruction and collection within the spinal cord.

Transverse myelitis T1-hypointensity/T2-hyperintensity corresponding to demyelination lesions, that commonly extended for 3-4 spinal segments Poliomyelitis Bilateral T2-hypersignal in the anterior horn of the spinal cord.

HTLV-I associated

Cord swelling and high intensity in the central myelopathy portion along the spinal cord in T2-sagittal images Neuromyelitis optica T1-hypointensity/T2-hyperintensity extended for more than 3 vertebral bodies associated with a central grey matter involvement and cord atrophy.

Dorsal root ganglionopathy T2-hyperintensity at the dorsal column and cord atrophy.

Spinocerebellar ataxia

Spinal cord atrophy.

Research setting

In the research setting, more advanced MRI techniques are used to image the spinal cord. In

Advanced MRI

The spinal cord is a common site for development of several MND that are mainly represented by ALS and SMA. Furthermore, the spinal cord can be affected by inflammatory diseases such as multiple sclerosis (MS) and by spinal cord injuries (SCI). In all of these disorders, the involvement of the spinal cord reflects a combination of several microscopic factors including axonal degeneration, demyelination or motoneurons loss. All these diseases represent challenges for diagnosis, prognosis, follow-up and monitoring of the effect of investigational drugs.

The present section focused on advanced MRI techniques that are currently used to quantify spinal cord white and grey matter integrity in the pathological spinal cord and particularly in MND: i) highresolution anatomic data to visually identify the extent of the spinal cord lesions and more importantly to infer on white and grey matter atrophy; ii) DTI and MT imaging to infer on axonal degeneration, loss and demyelination.

Spinal cord atrophy quantification from MR images

Common atrophy indexes

In spinal cord disorders, axons and/or motoneurons loss can be assessed in vivo indirectly from MR images by quantifying spinal cord atrophy at a given cord level. The estimation of spinal cord crosssectional area at given level was, for more than a decade, the most used approach to investigate atrophy in the pathological context, mainly in MS, that represents the main body of the scientific research on spinal cord pathologies. The cross-sectional approach consists of estimating a mean cord surface over a representative number of slices at a given vertebral level, typically five continuous thick slices perpendicular to the spinal cord at C2 vertebral level (Losseff et al., 1996). There were three main reasons that explain such a choice of vertebral level: i) the high contrast between the CSF and the spinal cord, as well as the large CSF space, low cross-sectional area variability, low cerebrospinal fluid flow speeds and less cord motion, which increases atrophy quantification methods' reproducibility and accuracy at this level using a variety of magnetic strength, coils and MRI sequences; ii) (Agosta et al., 2009), showed a significant cord area change between the baseline and a mean follow-up of 9 months (17 ALS patients; p = 0.003) with no correlations with the ALSFRS changes. Alternatively, some studies proposed a variety of indexes derived from cross-sectional approach (Figure 12):

-Cord anterior-posterior width (APW) and left-right width (LRW) (Lundell et al., 2011). These indexes provide, in the pathological context, information on cord flattening.

-Cord eccentricity (CE), which is defined as the square root of 1 -(APW/LRW) 2 . CE index provides, in the pathological context, information on cord flattening [START_REF] Fahl | Spinal cord damage in Machado-Joseph disease[END_REF]Branco et al., 2014).

-Cord radial distance, which is defined as the angular variation of the spinal cord radius over the full circle (Lundell et al., 2011).

The eccentricity index was used in the context of sporadic ALS and spinocerebellar ataxia type 3.

No cord flattening was found in ALS patients (43 patients versus 43 age and gender-matched healthy controls) (Branco et al., 2014). In spinocerebellar ataxia type 3 patients (48 patients versus 48 age and gender-matched healthy controls), there was a significant antero-posterior cord flattening (p = 0.005), with no correlation with the clinical scale for assessment and rating of ataxia [START_REF] Fahl | Spinal cord damage in Machado-Joseph disease[END_REF].

Cord widths and radial distance indexes were used in the context of chronic SCI (19 patients versus 16 age and gender-matched healthy controls) (Lundell et al., 2011). The authors showed a preferential anterior-posterior cord atrophy at C2 vertebral level or cord flattening as consequence of the cord trauma. In SCI patients, the APW and the LRW were exclusively correlated with sensory scores and motor score, i.e. light touch and pinprick for sensory scores, and muscle strength for motor score) (p # 0.01), consecutively. Radii in the anterior and posterior parts of the spinal cord were correlated to sensory scores (p < 0.01) (Figure 13). Radii in the lateral and ventro-lateral parts were correlated to motor score (p < 0.01) (Figure 13). The anterior and the posterior parts of the spinal cord represent the main ascending sensory pathways, and the lateral and ventro-lateral parts of the spinal cord represent the main descending motor pathways and motoneurons. This study was the first to propose a focal atrophy investigation of spinal cord sensory-motor systems at C2 vertebral level. This study represents one of the most innovative approaches to quantify cord atrophy because of the intuitiveness and simplicity of its method as well as its potential generalization to explore specifically white matter sensory and motor pathways atrophy as well as motoneurons loss in large regions of the spinal cord in MND.

Thanks to the recent improvement in MRI technology, scanners with higher magnet strength and the large number of phased-array coils, as well as the gained technical/methodological knowledge in imaging the spinal cord, lead to feasible picturing of large spinal cord regions, i.e. cervical and upper thoracic spinal or the whole thoracic and lumbar spinal cord, with high resolution, SNR and CNR (typically 3D sequences with voxel size ≤ 1 mm 3 ) or white and grey matter tissues at specific spinal cord levels with high resolution (typically 2D sequences with thick slices 4-5 mm, and in plane resolution ≤ 0.5×0.5 mm 2 ) (Figure 7). 14). The authors showed differences in the atrophy profile between MS phenotypes, i.e. CIS, PPMS, benign MS, SPMS and RRMS. In these two studies, VBM enabled a better characterization of the clinical heterogeneity in MS patients. However, the latter studies have a major limitation, which is their inability to study regional white and grey matter atrophy, which would require the use of higher MR imaging resolution sequences with an optimal contrast between white and grey matter. Despite this, VBM could be of interest in characterizing regional atrophy in MND, i.e. loss of spinal motoneurons, as well as the clinical heterogeneity in MND and particularly in ALS.

As previously mentioned, the new generation of MR scanners and coils enables the production of high resolution MR images that allow picturing white and grey matter tissues at specific spinal cord levels, thus a specific tissue atrophy quantified in spinal cord diseases. There are mainly two ways to process these MR images: i) by estimating a mean white and grey matter areas within a representative number of slices at a given cord level, similar to the cross-sectional area measurement; ii) delineating white and grey matter tissues then using them as inputs in an advanced morphological analysis method based on structures shape, i.e. TBM. The first approach was used in two major impact MS studies [START_REF] Schlaeger | Spinal cord gray matter atrophy correlates with multiple sclerosis disability[END_REF][START_REF] Schlaeger | Association Between Thoracic Spinal Cord Gray Matter Atrophy and Disability in Multiple Sclerosis[END_REF]. In the first study, white and grey matter areas were measured at the disc levels C2/C3, C3/C4, T8/9, and T9/10 in 142 MS patients (99 relapsing and 43 progressive MS patients) and 20 healthy controls. The authors showed thoracic grey matter atrophy with absence of white matter atrophy in relapsing MS patients. Grey matter atrophy was more pronounced in progressive MS than in relapsing MS and correlated with clinical disability and lower limb function.

Cervical grey matter areas had the strongest correlation with clinical disability followed by thoracic cord grey matter area and brain grey matter volume. More interestingly, their study showed the presence of grey matter atrophy at the cervical and thoracic levels, which may reflect a diffuse grey matter degeneration along the whole spinal cord. This method was proposed, for the first time, to map spinal cord white and grey matter alterations occurring with age (Taso et al., 2015). The authors showed that regional spinal cord atrophy was mostly localized in the anterior horns in elderly healthy subjects (n =25) compared to young ones (n = 40) (the spinal cord was imaged at the middle of C1 to C6 vertebral bodies) (Figure 15). This result is consistent with in-vivo imaging and post-mortem studies described above (Valsasina et al., 2012;[START_REF] Moral | Evaluation of neuronal loss, astrocytosis and abnormalities of cytoskeletal components of large motor neurons in the human anterior horn in aging[END_REF][START_REF] Morrison | The Effect of Advancing Age upon the Human Spinal Cord[END_REF]. This method holds great promise for investigating motoneurons loss occurring in MND.

Axonal degeneration, loss and demyelination quantification from diffusion-weighted

and magnetization transfer imaging.

Diffusion-weighted imaging

Diffusion results from the random movement of molecules in-vivo. Diffusion of water in structures such as the CSF or grey matter is isotropic, i.e. identical in all directions, whereas in white matter it occurs preferentially along the axis of orientation of the fiber bundles. Diffusion tensor imaging allows the measurement of water diffusion anisotropy along and within white matter axons (Yang et [START_REF] Pierpaoli | Toward a quantitative assessment of diffusion anisotropy[END_REF]. Water diffusion in the tensor model is presumed to be Gaussian and unidirectional.

Diffusion tensor (D) is 3"3 matrix that describes tissue anisotropy at each image voxels:

! ! ! !! ! !" ! !" ! !" ! !! ! !" ! !" ! !" ! !!
Diffusion tensor decomposition to principal diffusion magnitudes and directions results in ordered three eigenvalues (" 1 , " 2 , " 3 ) and eigenvectors (V 1 , V 2 , V 3 ). The eigenvalue " 1 represents the diffusion magnitude along V 1 , i.e. along fiber bundles, and λ 1 and λ 2 the diffusion magnitude along V 2 and V 3 , respectively, i.e. on the plane perpendicular to fiber bundles. Therefore, diffusion anisotropy indexes that characterize different aspects of water diffusion distribution at each image voxel, i.e. ellipsoid, can be computed from those eigenvalues, regardless of directionality. Principal diffusion indexes are mean diffusivity (MD), fractional anisotropy (FA), axial and radial diffusivities (AD and RD):

!" = ! ! + ! ! + ! ! 3 !" = 3 2 (! ! -!") ! + (! ! -!") ! + (! ! -!") ! ! ! ! + ! ! ! + ! ! ! !" = ! ! !" = ! ! + ! ! 2
The model behind DTI, which represents water diffusion mainly along and perpendicular to fiber bundles planes, is simple and shows its limit in representing the complex diffusion processes occurring, for example, in non-orthogonal crossed fibers or multiple fibers orientations (Campbell et indexes and white matter microstructures should carefully be interpreted. Therefore, we can only conclude that diffusion indexes, whether estimated using tensor or q-ball models, allow a general evaluation of white-matter pathways integrity. For an exhaustive description of the correlations between diffusion indexes and white matter tissue microstructures, the reader is referred to the review from Concha (2014). RD was negatively correlated with the ALSFRS-R, finger and foot tapping speed (p < 0.05) as well as the respiratory function (p < 0.01), as measured by the force vitality function scale (Nair et al., 2010).

In the longitudinal study from Agosta et al. (2009) (17 ALS patients versus 20 age-matched controls), ALS patients showed a significant longitudinal decrease in FA, and an increase in MD at the cervical spinal cord level (p = 0.01). Longitudinal change of diffusivity indexes did not correlated with the ALSFRS changes. For an exhaustive description of DTI applications in spinal cord diseases, the reader is referred to the review from Martin et al. (2013).

Magnetization transfer imaging

Hydrogen nuclei linked to macromolecules such as proteins and lipids constituted of axons myelin sheet (70-80% of lipids and 20-30% of proteins), have an extremely short T2 signal (~10-5 s). These macromolecules are not directly detectable with standard MRI sequences. First, macromolecular spins are saturated using an off-resonance RF pulse, then transfer of magnetization between bound and free pools occurs by means of cross relaxation processes (dipole-dipole interactions, chemical exchange, etc.). This transfer to the « free » pool induces a signal decrease that can be indirectly measured, known as MT imaging [START_REF] Filippi | Magnetization transfer magnetic resonance imaging of the brain, spinal cord, and optic nerve[END_REF][START_REF] Smith | Pulsed magnetization transfer imaging with body coil transmission at 3 Tesla: feasibility and application[END_REF]Wolf and Balaban, 1989). The latter interaction and/or exchange can be quantified using a magnetization transfer ratio (MTR), which is calculated as the percentage difference of MT images with macromolecules signal saturation (M S ) and one without (M 0 ):

!"# = 100× ! ! -! ! ! !
The MTR enables inferring on myelin content, axonal count and density as shown by three MS histological studies at the brain and spinal cord levels, and thus to assess demyelination/remyelination and degeneration [START_REF] Schmierer | Quantitative magnetization transfer imaging in postmortem multiple sclerosis brain[END_REF][START_REF] Schmierer | Magnetization transfer ratio and myelin in postmortem multiple sclerosis brain[END_REF][START_REF] Mottershead | High field MRI correlates of myelin content and axonal density in multiple sclerosis--a post-mortem study of the spinal cord[END_REF].

Objectives

The spinal cord represents the entry for sensory information and the exit for motor commands for the limbs and the trunk. Lesions of the spinal cord due to trauma or neurodegenerative diseases thus lead to major sensory and motor consequences whose severity depends on the extent of the lesions. It is therefore extremely important to establish the extent of the spinal lesions as precisely as possible by using several complementary approaches to clinical and electrophysiological examinations such as imaging of the spinal cord, which was the main topic of the SPINE 1 Imaging Project.

Our research group significantly contributed to the development of a multi-parameter MRI approach to studying spinal cord diseases, particularly MND, which was achieved in the context of the

Results

Methodological studies

Study 1 -Validation of a semi-automated spinal cord segmentation method Study 2 -Fast and accurate semi-automated segmentation method of spinal cord MR images at 3T applied to the construction of a cervical spinal cord template This method has to be initialized manually by drawing two contours, one to delineate the spinal cord and the other surrounding the CSF. However, this method has only been validated at C2 vertebral level using 1.5T images in MS patients [START_REF] Horsfield | Rapid semi-automatic segmentation of the spinal cord from magnetic resonance images: application in multiple sclerosis[END_REF]Losseff et al., 1996).

Multi-parametric MRI studies in MND

To validate cord atrophy measurement at 3T using Losseff's segmentation method, we evaluated its reproducibility, accuracy and robustness to manual initialization at both the cervical and thoracic levels and in neurological disorders other than MS.

Material and methods

A large dataset of 111 subjects including 49 healthy subjects, 29 ALS, 19 adult SMN1 gene-linked SMA and 14 SCI patients were enrolled in the study. Losseff's segmentation method was used to measure the spinal cord area from high-resolution T2-weighted 3D turbo spin echo images (52 sagittal slices, FOV=280 mm, voxel size=0.9×0.9×0.9 mm 3 , acquisition time ~6 min), in the cervical and thoracic spinal cord (from C2 to T9). Method reproducibility and accuracy were evaluated. The robustness of the method to initialization was assessed by simulating modifications to the contours drawn manually prior to segmentation.

Results synthesis

This study showed the high reproducibility and accuracy of the semi-automated method from Losseff et al. (1996) for measuring a cross-sectional area of the spinal cord in both cervical and thoracic regions at 3T in a large number of normal subjects and a variety of patients with spinal cord disorders (Table 2). Improved image spatial resolution obtained at 3T allowed higher segmentation performances than reported previously (Losseff et al., 1996;[START_REF] Horsfield | Rapid semi-automatic segmentation of the spinal cord from magnetic resonance images: application in multiple sclerosis[END_REF]. By conducting numerical simulations, we have shown that method was highly robust to its initial contours. More specifically, simulations suggested optimal segmentation results would be obtained with initial contours representing as little as 10% to 30% of manually drawn areas for both the spinal cord and the CSF (Figure 2).

Therefore, full automation of the method could be envisaged by automatically extracting CSF and cord regions, while allowing large range of error rate in contours extraction. Finally, as the method was efficient at different levels and in various diseases of the spinal cord, it is expected that it may be applied in a wide range of neurological diseases for a reproducible and accurate quantification of spinal cord atrophy. The high performances of the Losseff's method make us confident about the accuracy of spinal cord atrophy quantification in the context of MND (Study 4).

Erratum:

In Table 1, the French abbreviation SLA was used instead of ALS.

Study 2 -Fast and accurate semi-automated segmentation method of spinal cord MR images at 3T applied to the construction of a cervical spinal cord template

Background and objectives

In Study 1, we showed that the semi-automated method by Losseff et al. (1996) demonstrated high reproducibility and accuracy for measuring spinal cord cross-sectional area in both cervical and thoracic regions at 3T in normal subjects and in patients with MND and SCI. Improved image spatial resolution obtained at 3T allowed higher segmentation performances than reported previously. More interestingly, full automation of the method seems possible given its low sensitivity to the initialization steps. Losseff's segmentation method is a semi-automated method, and thus needs a large manual intervention from an operator, which restrains its use to quantify atrophy in few representative slices of the cord region of interest.

In clinical practice, it is often difficult to visually determine spinal cord atrophy. The growing interest in assessing spinal cord atrophy using MRI therefore underlines the need for a fast and accurate segmentation method of the spinal cord with limited manual intervention. This method would also enable segmentation of large data sets in a limited time, thus facilitating investigating in large cord regions using either volume or cross-sectional area measurements. Particularly, this method would enable investigation of the topography of spinal cord atrophy in spinal cord diseases using morphometry methods (Morra et al., 2009;Wang et al., 2010Wang et al., , 2011)). Furthermore, to be used, advanced processing methods require an additional step of data normalization. The idea behind data normalization is to adjust for intersubject variability that is not related to the studied pathophysiological process [START_REF] Mazziotta | A probabilistic atlas and reference system for the human brain: International Consortium for Brain Mapping (ICBM)[END_REF], as well as to realign all data in the same reference, i. This second study was designed to address two main objectives: i) to construct and validate a new fast and accurate segmentation method with minimal manual intervention benefitting from the high robustness of Losseff's method to its initialization procedure; ii) as an application, we extended the existing work at 1.5 and 3T (Fonov et al., 2014;Valsasina et al., 2012) to data normalization and the construction of an accurate MRI template of the cervical spinal cord using a large sample of 3T images.

Material and methods

A semi-automated double threshold-based method (DTbM) was proposed enabling both crosssectional and volumetric measures from 3D T2-weighted turbo spin echo MRI scans of the spinal cord at 3T (52 sagittal slices, FOV=280 mm, voxel size=0.9×0.9×0.9 mm 3 , acquisition time ~6 min). Eightytwo healthy subjects, 10 patients with ALS, 10 with adult SMN1 gene-linked SMA and 10 with SCI were studied. The DTbM was compared with active surface method (ASM) [START_REF] Horsfield | Rapid semi-automatic segmentation of the spinal cord from magnetic resonance images: application in multiple sclerosis[END_REF]) and

Losseff's segmentation method (Losseff et al., 1996), two of the most used segmentation methods to quantify atrophy (De leener et al., 2016a), taking manual outlining from an experienced operator (with four years' experience) as the ground truth. An independent expert neuroradiologist (with seven years' experience) visually scored DTbM and ASM segmentations in cervical and thoracic cord regions. The operator was blinded to the type of data and methods. Accuracy was also quantified at the cervical and thoracic levels as well as at the C2 vertebral level. Fifty-nine MR images from healthy subjects were used to construct a cervical spinal cord template and a standardization pipeline was designed.

Results synthesis

In this second methodological study we designed a segmentation method with minimal manual intervention as well as a data normalization pipeline. Visual scoring showed better performance for DTbM than for ASM in both healthy and pathological groups (Table 3). More importantly, the neuroradiologist did not find any visual abnormalities in most cases of ALS and SMA data, justifying the need for quantitative methods to analyze MND data, which represented one of my main scientific focus during my thesis. Quantitative accuracy analysis showed that DTbM outperformed ASM at both cervical and thoracic spinal cord levels as well as at C2 vertebral level in both healthy and pathological groups, confirming the visual evaluation results (Tables 4 and5). Our method showed similar accuracy compared with Losseff's method, but with the advantage of limited manual interaction. An accurate data normalization pipeline was designed that included DTbM, enabling the construction of an accurate straight cervical spinal cord template and a tissue probability map (Figure 3). Our segmentation method may also accurately measure cross-sectional area and spinal cord volume in a large range of pathologies.

More interestingly, the proposed tools could be used to investigate the topography of spinal cord atrophy in spinal cord diseases using morphometry methods (Morra et al., 2009;Valsasina et al., 2012Valsasina et al., , 2013;;Rocca et al., 2013). Among these methods, surface-based morphometry was used to map cord atrophy in the context of adult SMN1-linked SMA (Study 5).

Fast and Accurate Semi-Automated Segmentation Method of Spinal Cord MR Images at 3T Applied to the Construction of a Cervical Spinal Cord Template

Materials and Methods

A semi-automated double threshold-based method (DTbM) was proposed enabling both cross-sectional and volumetric measures from 3D T 2 -weighted turbo spin echo MR scans of the spinal cord at 3T. Eighty-two healthy subjects, 10 patients with amyotrophic lateral sclerosis, 10 with spinal muscular atrophy and 10 with spinal cord injuries were studied. DTbM was compared with active surface method (ASM), threshold-based method (TbM) and manual outlining (ground truth). Accuracy of segmentations was scored visually by a radiologist in cervical and thoracic cord regions. Accuracy was also quantified at the cervical and thoracic levels as well as at C2 vertebral level. To construct a cervical template from healthy subjects' images (n=59), a standardization pipeline was designed leading to well-centered straight spinal cord images and accurate probability tissue map.

Results

Visual scoring showed better performance for DTbM than for ASM. Mean Dice similarity coefficient (DSC) was 95.71% for DTbM and 90.78% for ASM at the cervical level and 94.27% for DTbM and 89.93% for ASM at the thoracic level. Finally, at C2 vertebral level, mean DSC was 97.98% for DTbM compared with 98.02% for TbM and 96.76% for ASM.

Introduction

Spinal cord atrophy is a neuroimaging feature that is associated with motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) [1][2][3][4][5], inflammatory diseases such as multiple sclerosis (MS) [6][7][8], and spinal cord injuries (SCI) [9][10][11].

In the past two decades, several segmentation methods with varying degree of manual intervention have been proposed to assess cord atrophy from MR images [12][13][14][15][16][17][18][19]. These methods have been validated using mostly 1.5T images and mainly investigated cord atrophy in MS patients, at the upper cervical level where the spinal cord is most atrophied [20]. In the context of spinal cord diseases, there is clear interest in investigating larger portions of the spinal cord. Indeed, degenerative diseases of the spinal cord, such as ALS and SMA, are characterized by diffuse degeneration of lower motor neurons localized in the anterior horn of all spinal cord levels [21,22]. The entire corticospinal tract also degenerates in ALS [22]. Spinal cord injuries can also affect all spinal cord levels [23].

In clinical practice, it is often difficult to visually determine spinal cord atrophy. The growing interest in assessing spinal cord atrophy using MRI therefore underlines the need for a fast and accurate method of spinal cord segmentation with limited manual intervention. Such method would indeed facilitate the investigation of large regions of the spinal cord using either volume or cross-sectional area (CSA) measurements. This method would also enable segmentation of large data sets in limited time. Furthermore, this would open the way to the construction of a template that could be used for cord normalization in group analyses. Several methods have been proposed to construct a template of the cervical spinal cord [24][25][26]. In [24,26], less than 20 subjects were used to construct a T2-weighted 3T MRI template and such small sample was not representative enough of the anatomical variability of the spinal cord. In [24], a large amount of manual intervention was needed to straighten the spinal cord (one had to draw manually a line along the anterior edge of the spinal cord for each subject), while we introduce in this paper a segmentation method with minimal manual intervention. The cord straightening approach proposed in [24] leads to a better alignment of the anterior cord regions than the posterior cord regions, which is not the case in the present study as we use the cord centerline to straighten the spinal cord. Recently, a T1-weighted 1.5T MRI template has been proposed for the cervical spinal cord to quantify age-related atrophy as well as disease-related atrophy in MS patients [25,27,28]. However the authors used low-resolution 1.5T images and a semi-automated method, namely the active surface method (ASM) [14], which requires important manual intervention and lacks accuracy (CSA was reported to be overestimated by approximately 14% at C2 vertebral level compared with manual outlining).

In previous studies, the semi-automated threshold-based method (TbM) proposed in [18] proved highly accurate and reproducible at 1.5T (i.e., inter-, intra-operator and scan-rescan reproducibility) [14,18,29]. Recently, TbM has been validated at 3T in a large dataset of 111 subjects including healthy volunteers, ALS, SMA and SCI patients [30]. TbM underestimated CSA by 3.29% at the cervical level and 1.70% at the thoracic level. Interestingly, TbM proved highly robust to initialization suggesting potential automation.

The present study therefore has two main objectives: 1) To construct and validate a new fast and accurate segmentation method with minimal manual intervention benefitting from the high robustness of TbM to its initialization procedure; 2) As an application, to extend existing work at 1.5 and 3T [25,26] to the construction of an accurate MRI template of the cervical spinal cord using a large sample of 3T images.

Materials and Methods

MRI acquisition

Subjects were positioned head-first supine, with a 2-centimeter-thick pillow to lift the head and no pillow below the neck. This strategy was used to limit the natural cervical cord lordosis at around C3-C4, i.e., excessive cord curvature in the antero-posterior direction. Subjects were positioned as comfortably as possible, centered in the scanner's reference, and were systematically asked not to move during the acquisition in order to minimize motion artifacts.

Scans were performed at CENIR, Pitié-Salpétrière Hospital, Paris, France, using a 3T MRI system (TIM Trio 32-channel, Siemens Healthcare, Erlangen, Germany). The spinal cord was imaged using a three-dimensional T2-weighted turbo spin echo (TSE) sequence with a slabselective excitation pulse (SPACE sequence: Sampling Perfection with Application optimized Contrasts using different flip angle Evolution). Imaging parameters were: voxel size = 0.9×0.9×0.9 mm 3 ; Field of View (FOV) = 280×280mm²; 52 sagittal slices; Repetition time (TR)/ Echo time (TE) = 1500/120ms; flip angle = 140°; generalized autocalibrating partially parallel acquisition (GRAPPA) with acceleration factor R = 3; turbo factor = 69; acquisition time ~6 min. This sequence provides high signal-to-noise ratio due to 3D acquisition, high resolution due to isotropic acquisition, short acquisition times by combining parallel acquisition with high turbo factors. No specific absorption rate limitation was observed in any of our acquisitions. In 1.5T spinal cord MRI studies, SPACE sequence showed an absence of signal loss due to pulsatile CSF motion and no artifacts due to swallowing or vessel pulsation [31], improving clinical diagnosis potential of MRI [32] and an excellent assessment of anatomy and resistance to artifacts in imaging difficult anatomy such as in scoliosis and spondylolysis [33], compared with conventional MRI sequences. At 3T, SPACE sequence showed an increased signal to noise ratio compared with 1.5T [34,35] and allowed better inter-observer agreement for detecting foraminal stenosis, central spinal stenosis, and nerve compression compared with 2D T2-weighted TSE sequence [36].

Segmentation method

Preprocessing. Data were corrected for B 1 non-uniformity intensity using Minc-Toolkit N3 [37]. Preprocessing of MR images using N3 has been shown to substantially improve the accuracy of anatomical analysis techniques such as tissue classification and segmentation as well as cortical surface extraction [38,39]. The operator had to choose a region of interest interactively using an in-house Matlab script (The Mathworks Inc., Natick, MA, USA), as follows:

The operator just had to display the sagittal slices where the spinal cord was the most median in the FOV, and to select with two mouse clicks the upper and lower limits of the spinal cord region to be segmented; the images were then automatically cropped to focus on this region of interest. This was the only manual intervention required in the whole segmentation procedure. The produced images were then resampled to a voxel size of 0.3×0.3×0.3 mm 3 using 3D cubic interpolation in order to maximize segmentation accuracy [11,30].

Segmentation: Volumetric approach. Conventional TbM [18] requires manual initialization since the operator has to delineate in each axial slice to be segmented the contours of the CSF and spinal cord regions. TbM then uses the mid intensity of the mean intensities in these regions as a threshold to classify voxels into two classes, namely spinal cord and CSF. By conducting numerical simulations, we have shown previously that TbM was highly robust to its initial contours [30]. More specifically, simulations suggested optimal segmentation results would be obtained with initial contours representing as little as 10% to 30% of manually drawn areas for both spinal cord and CSF. Full automation of the TbM method could therefore be envisaged by automatically extracting CSF and cord regions, while allowing 10% to 30% error rate as compared with manually drawn areas. This led us to design a double threshold-based method with minimal manual intervention (DTbM) implemented using Matlab, which involved the following steps in each slice of the region of interest to be segmented (see Fig. 1 for a flowchart of the method): S1: The slice was segmented using Otsu's global thresholding method (first threshold of DTbM) [40]. This method seeks for the optimal grey level threshold that maximizes the interclass variance between the foreground and the background of the MRI images. The optimal threshold enables to select pixels of highest intensity, corresponding mainly to CSF. S2: A binary mask M CSF of the CSF was obtained by selecting from these pixels the cluster with the highest intensity and a minimum size of 50 connected pixels.

S3: A flood-fill algorithm [41] was applied in order to fill the inner part of M CSF , yielding to a mask M F .

S4a: If M F -M CSF was zero, this meant that the border of the CSF mask was not completely closed and there was no inner region to fill. The global contrast of the initial MRI slice was then enhanced by truncating the image histogram by 0.1% of its upper end and then steps S1 to S3 were repeated until M F -M CSF was not zero, which meant that the border of the CSF mask was closed and the inner part had been completely filled.

S4b: If M F -M CSF was not zero, this difference between M F and M CSF corresponded to the inner part of the CSF mask, i.e. a binary mask of the spinal cord (M SC ).

S5: M SC and M CSF were eroded (disk radius of 3 pixels) in order to lie between 10% to 30% error rate as compared with the expected manually drawn areas [30].

S6: CSF and spinal cord regions obtained by masking the initial image by the binary masks from S5 were taken as initial inputs to the threshold-based method (TbM) proposed by Losseff et al. [18], as demonstrated in [30]. TbM uses the mid intensity of the mean intensities in the two regions as a threshold (second threshold of DTbM) to further classify region pixels into two classes, namely the spinal cord and the CSF [18]. CSA was defined as the mean area in mm 2 of the resulted masks. S7: The segmented spinal cord regions obtained from S6 in all slices were corrected in order to adjust for possible missing or inaccurate segmentations. First, the centerline of the spinal cord was extracted by locating the center of mass of the spinal cord region in each slice. The centerline curve was smoothed using robust locally weighted regression [42]. The derivative of the centerline and polynomial curve fitting were used to detect aberrant discontinuities in the centerline then to remove them (a threshold of 15 voxels was set). Missing points in the centerline were then estimated using cubic spline interpolation. The radius from the center of mass of each axial mask to its borders was measured with an angular resolution of 5°by mapping Cartesian coordinates to polar coordinates (radius every 5°) [11]. To limit nerve roots contribution to the final segmentation, the length of the radiuses was smoothed for each angle using robust locally weighted regression across slices. Then, radiuses of the missing masks were estimated for each angle using cubic spline interpolation across slices. Finally, the contours of the spinal cord were calculated by converting polar coordinates (center of mass and radius every 5°) back to Cartesian coordinates. Finally, contours were converted to binary masks leading to a 3D mask covering the whole spinal cord region.

Segmentation: cross-sectional area approach. When only a few slices needed to be segmented, the volumetric approach was replaced by a cross-sectional area approach, which merely consisted of segmenting preprocessed images using steps S1 to S6 as described above.

Comparison with existing methods

First, the volumetric measures obtained from DTbM in all spinal cord vertebral levels were compared with those obtained from ASM [14] using visual scoring. Second, the CSA measures obtained from DTbM at C2 vertebral level were compared with those obtained from ASM and TbM [18] using quantitative criteria, taking manual outlining as the ground truth. All segmentations were achieved by an experienced operator (i.e. four years' experience) using in-house Matlab scripts for manual outlining, DTbM and TbM and a trial version of Jim 6.0 (Xinapse Systems, UK; http://www.xinapse.com) for ASM. ASM is based on image smoothing followed by an active surface model constrained by intensity gradient image information. The operator placed landmarks on the center of the spinal cord in the axial plane at the extremes of the cord region to be segmented and (i) every 10 axial slices for volumetric approach and (ii) in all axial slices for CSA measures. The parameters of the method were: nominal cord diameter: range = 7-10 mm; number of shape coefficients = 24; order of the longitudinal variation = 12. For parameter adjustment, the experienced operator was guided by the user's manual of the method (http://www.xinapse.com/Manual/), data characteristics as well as visual assessment of segmentation results.

Subjects

One hundred and twelve subjects were randomly selected from our database of spinal cord 3T MRI scans. There were 82 healthy volunteers, 10 patients with ALS, 10 patients with SMA and 10 patients with SCI. Four healthy subjects were excluded from our study due to disk protrusion compressing the spinal cord (n = 2, at C5/C6 level) or inducing loss of CSF space and contacting the spinal cord (n = 2, at C3/C4, C4/C5 and C5/C6 levels). The local Ethics Committee of our institution approved all experimental procedures (Paris-Ile de France Ethical Committee under the 2009-A00291-56 registration number), and written informed consent was obtained from each participant. MRI experiments were conducted according to the principles expressed in the latest revision of the Declaration of Helsinki.

Evaluation

Groups. MR images from the healthy subjects comprised two fields of view, one covering the whole cervical and the upper thoracic spinal cord regions (n = 60) and the other including partly the cervical and the thoracic spinal cord regions (n = 18). MR images from the patients covered the whole cervical and upper thoracic spinal cord regions. Therefore, subjects were split into two groups:

• Group 1 included 7 subjects randomly chosen from the 60 healthy volunteers with images covering the whole cervical and the upper thoracic spinal cord, 3 subjects randomly chosen from the 18 healthy volunteers with images including partly the cervical and the thoracic spinal cord (mean age of selected healthy subjects ± SD: 41.9 ± 16.5 years, 4 females), 10 patients with ALS (mean age ± SD: 54.5 ± 10.9 years, 3 females), 10 patients with SMA (mean age ± SD: 36.1± 10.9 years, 8 females), and 10 patients with SCI (mean age ± SD: 46.4±18.2 years, 2 females). Table 1 shows the segmented spinal cord region for each subject. Their images were used for visual evaluation of DTbM and ASM segmentations using the volumetric approach at the cervical and thoracic spinal cord regions. They were also used for quantitative evaluation of CSA accuracy measured at the middle of each available spinal cord vertebral level (Table 1).

• Group 2 included 10 healthy volunteers randomly chosen from the 60 healthy subjects with images covering the whole cervical spinal cord (mean age ± SD: 45.7 ± 15.8 years, 6 females), and the same ALS, SMA and SCI patients as in Group 1. Their images were used for quantitative comparison of DTbM, TbM and ASM segmentations at C2 vertebral level. This region had high contrast between CSF and spinal cord as well as large CSF spaces and low CSA variability, and therefore segmentation was expected to be more reproducible and accurate at this level. Furthermore, recent studies measuring CSA have shown pronounced cord atrophy at C2 vertebral level in ALS and SCI patients that correlates with clinical disability [1,10,11].

All data were preprocessed using the method described in the "Preprocessing" section above.

Visual evaluation (volumetric approach). Images from Group 1 were segmented using the volumetric version of DTbM (steps S1 to S7) and ASM [14]. Accuracy of DTbM and

C2-T5 C2-T2 C2-T5 C2-T4 C7-T9 C2-T3 C2-T4 C2-T3 C2-T4 C2-T5 C2-T6 C2-C7 C3-T8 C2-T4 C2-T5 C2-T3 C2-T1 C2-T1 C2-T6 C2-T1 C3-T8 C2-T5 C2-T6 C2-T3 C2-T4 C2-T5 C2-T5 C2-T4 C2-T3 C2-T5 C2-T5 C2-T2 C2-T4 C2-T6 C2-T4 C2-T4 C2-T6 C2-T4 C2-T5 C2-T4
doi:10.1371/journal.pone.0122224.t001

ASM segmentations was evaluated visually by an independent expert neuroradiologist (with seven years' experience) using the OsiriX Imaging Software (http://www.osirix-viewer.com/). The operator was blinded to the type of data and methods. The evaluation used a combined score based on neuroradiological diagnosis and overlap criteria (Table 2): The operator evaluated visually the overlap between the spinal cord region and the mask resulting from each segmentation method in the axial plane, in the whole spinal cord in the FOV (Table 1, global overlap) and in specific regions of the spinal cord that were subject to T 2 -hyperintensity, atrophy, or narrow spinal canal, which are usually present in clinical routine MR images of the pathological spinal cord. The operator also evaluated segmentation accuracy in visually normal appearing spinal cord regions and nerve roots regions. The overlap criteria were:

• Insufficient: The method failed to segment accurately the spinal cord. The resulted axial masks underestimated/overestimated by a large number of voxels the visually expected masks.

• Moderate: The resulted axial masks were close to but partially larger or smaller than the expected spinal cord region.

• Substantial: The resulted axial masks underestimated/overestimated the visually expected spinal cord region by only few voxels.

• Perfect: Resulted axial masks fitted perfectly the spinal cord region.

• Included: Resulted axial masks included voxels from nerve roots.

• Not included: Resulted axial masks did not include voxels from nerve roots.

Quantitative evaluation. Quantitative evaluation of CSA measures obtained on the whole spinal cord using the volumetric approach would have required manual segmentation of a large number of slices to get a ground truth, which was untractable. Nevertheless, to try and evaluate the performance of the volumetric approach at all cord levels, a ground truth was obtained by manually segmenting the middle slice of each available spinal cord vertebral level (Table 1) for all subjects of Group 1. In the SCI group, only vertebral levels rostral and caudal to the spinal cord lesions were considered [10,30]. CSA values obtained in this middle slice using DTbM (see section "Segmentation: volumetric approach" above) and ASM were compared with manual outlining.

On the other hand, we carried out a specific evaluation of the cross-sectional approach at C2 vertebral level using images from Group 2, in order to conform to segmentation evaluations largely conducted in the literature. This required straightening the spinal cord before segmentation, as follows: the cord centerline was extracted (i.e. the line linking the centers of mass of all axial masks) and further smoothed using robust locally weighted regression. Spinal cord images were resampled in planes perpendicular to the centerline using 3D cubic interpolation leading to straight spinal cord images. Five three-millimeter-thick slices were selected with the most inferior slice passing centrally through the C2/C3 disk, as largely made in MS and SCI studies at C2 vertebral level [7-10, 14, 18, 43-46]. Unlike what has been proposed in [16,43], we chose to resample data to account for partial volume between the spinal cord and the CSF as proposed in [11,30]. In addition, cord straightening enabled to reduce partial volume due to the cord orientation between the CSF and the spinal cord as well as blurring at the interface between the two tissues, and thus increase segmentation accuracy [16,43]. Images were segmented using manual segmentation, TbM, DTbM (as described in "Cross-sectional area approach" section above), and ASM. Accuracy was evaluated in two ways: (1) by using the Dice similarity coefficient (DSC), which evaluates the performance of segmentation methods by measuring their spatial overlaps [47]; the DSC is defined as follows: 2×|GT \ S| ⁄ (|GT| + |S|), where |GT| and |S| are the CSA in mm 2 obtained by the manual segmentation (ground truth) and the segmentation method to be evaluated (DTbM, TbM or ASM), respectively. |GT \ S| is the area in mm 2 common to both segmentation results; (2) by computing the relative CSA estimation error made by DTbM, TbM and ASM, considering the CSA from manual segmentation made by the experienced operator as the ground truth.

Template construction

The 60 healthy subjects with images covering the whole cervical spinal cord (mean age ± SD: 44.3 ± 16.2 years, range: 21-72 years, 31 females) were used to construct the cervical spinal cord template.

Preprocessing. The Minc-Toolkit N3 tool [37] was used to correct MR images for intensity non-uniformity then to apply intensity normalization. The experienced operator defined manually two landmarks in the sagittal slice where the spinal cord was the most median in the FOV to delimit the cervical spinal cord (i.e. from the upper limit of C2 vertebral level to the middle vertebral body C7/T1). After cropping and resampling the images as described in the "Volumetric approach: preprocessing" section above, a second non-uniformity intensity correction was applied using N3 in order to remove residual artifacts.

Segmentation. The cervical spinal cord was segmented using the volumetric version of DTbM (steps S1 to S7) and the cord centerline was extracted (i.e. the line linking the centers of mass of all axial masks) and further smoothed using robust locally weighted regression.

Standardization. Spinal cord images and cord masks were resampled in planes perpendicular to the centerline using 3D cubic interpolation for cord images and 3D nearest neighbor interpolation for masks, the centerline being centered in the FOV, which avoided registration between individuals as done in [25]. Thereby, the spinal cord images were always centered in the antero-posterior and left-right directions. Resulting images were standardized in length by rescaling them to the median cord length using 3D cubic interpolation for cord images and 3D nearest neighbor interpolation for masks (median length across subjects = 402 slices) as proposed in [25].

Template and probability tissue map. Straightened cord images were averaged across subjects and smoothed using 3D Gaussian filter (FWHM = 0.3×0.3×0.6 mm) to insure the validity of the Gaussian random theory, as well as to correct for possible imperfections in the standardization process, as showed in [25], which yielded a template of the cervical spinal cord. Straightened cord masks were averaged, which yielded a tissue probability map for the spinal cord, where voxel value from 0 to 1 is the probability for the voxel to belong to the spinal cord.

Results

Statistical analyses were conducted with Matlab software. Wilcoxon signed-rank test was used to compare accuracy measurement of DTbM, TbM and ASM at C2 vertebral level, and DTbM and ASM at the cervical and thoracic levels; p values less than 0.05 were considered significant.

Computational time

The mean number (± SD) of segmented slices was 701±96 slices/subject. Computation time for the whole procedure was 3.9±1.2 min/subject (mean ± SD), which included preprocessing time: 38±4 s/subject, segmentation time: 1.9±1.1 min/subject, correction (step S7) time: 1.5 ±0.3 min/subject. This was achieved using a 64-bit Quad-core (Intel Xeon, processor speed: 2.67 GHz) workstation. Computation time was not optimized as the whole procedure was coded in Matlab language. Fig. 2 shows an example of segmentation result from DTbM in an ALS patient.

Visual evaluation (volumetric approach)

Accuracy results are shown in Table 3 for each group of subjects. DTbM outperformed ASM in both healthy and pathological groups for global overlap (number of scores for DTbM/ASM: 33/1 perfect, 7/31 substantial and 0/ 8 insufficient to moderate), overlap in normal appearing spinal cord (DTbM/ASM: 28/0 perfect, 11/8 substantial, 1/11 moderate and 0/21 insufficient) and nerve roots regions (DTbM/ASM: 39/36 not included and 1/4 included in the final segmentations). Both DTbM and ASM showed similar overlap performance in regions with T 2hyperintensity (DTbM/ASM: 6/4 substantial to perfect and 5/7 insufficient to moderate), atrophy (DTbM/ASM: 5/3 substantial to perfect and 4/6 insufficient to moderate) and narrow spinal canal (DTbM/ASM: 3/3 substantial to perfect and 3/3 insufficient to moderate). The individual visual scoring is shown in S1 Table.

Quantitative evaluation

Volumetric approach. Table 4 shows accuracy measurements at the cervical and thoracic levels. At the cervical level, the mean DSC value was 95.71% for DTbM and 90.78 for ASM (manual outlining was considered as the ground truth). At the thoracic level, the mean DSC value was 94.27% for DTbM and 89.93% for ASM. When considering separately each group of subjects, the mean DSC value was 96.07% for DTbM and 90.89% for ASM in the control group. In the ALS group, the mean DSC value was 95.71% for DTbM and 90.06% for ASM. In DTbM underestimated the mean CSA measured by manual segmentation (considered as the ground truth) by 4.86% and 6.49% and ASM overestimated the mean CSA measured by manual segmentation by 13.53% and 9.47% at the cervical and thoracic levels, respectively. When considering separately each group of subjects, DTbM underestimated the mean CSA measured by manual segmentation by 5.05% and 4.81% and ASM overestimated the mean CSA by 14.34% and 16.08% at the cervical and thoracic levels, respectively. In the ALS group, DTbM underestimated the mean CSA measured by manual segmentation by 4.38% and 6.87% and ASM overestimated the mean CSA by 15.44% and 14.99% at the cervical and thoracic levels, respectively. In the SMA group, DTbM underestimated the mean CSA measured by manual segmentation by 4.39% and 6.72% and ASM overestimated the mean CSA by 13.88% and 15.26% at the cervical and thoracic levels, respectively. Finally, in the SCI group, DTbM underestimated the mean CSA measured by manual segmentation by 5.61% and 7.54% and ASM overestimated the mean CSA by 11.08% and 12.08% at the cervical and thoracic levels, respectively. The behaviour of DTbM method is illustrated in S1 Fig.

Based on DSC values, DTbM showed a significantly higher accuracy for the whole population than ASM at both the cervical and thoracic levels (Wilcoxon signed-rank test, p<0.001).

Cross-sectional area approach at C2 vertebral level. Table 5 shows mean CSA measurements made by the experienced operator using manual outlining, as well as DTbM, TbM and ASM results. Results of accuracy are also given. The mean DSC value was high for DTbM (97.98%), TbM (98.02%) and ASM (96.76%). When considering separately each group of subjects, the mean DSC value was 98.18% for DTbM, 98.2% for TbM and 96.63% for ASM in the control group. In the ALS group the mean DSC value was 97.86% for DTbM, 97.92% for TbM and 96.83% for ASM. In the SMA group the mean DSC value was 98.01% for DTbM, 98.11% for TbM and 96.83% for ASM. Finally, in the SCI group, the mean DSC value was 97.86% for DTbM, 97.87% for TbM and 96.77% for ASM. DTbM and TbM underestimated the mean CSA measured by manual segmentation (considered as the ground truth) by 0.25% and 0.36%, respectively. ASM overestimated the mean CSA measured by manual segmentation by 5.02%. When considering separately each group of subjects, DTbM, TbM and ASM overestimated the mean CSA measured by manual segmentation by 0.34%, 0.10% and 6.21%, respectively, in the control group. In the ALS group, DTbM, TbM and ASM overestimated the mean CSA measured by manual segmentation by 0.34% for DTbM, 0.10% for TbM and 6.21% for ASM. In the ALS group, DTbM, TbM overestimated the mean CSA measured by manual segmentation by 0.25% for DTbM and 0.11% for TbM and 5.07%. In the SMA group, DTbM, TbM underestimated the mean CSA measured by manual segmentation by 0.63% for DTbM and 0.74% for TbM. ASM overestimated the mean CSA measured by manual segmentation by 4.66%. Finally, in the SCI group, DTbM, TbM underestimated the mean CSA measured by manual segmentation by 0.96% for DTbM and 0.91% for TbM. ASM overestimated the mean CSA measured by manual segmentation by 4.13%.

Based on DSC values, DTbM showed a significantly higher accuracy for the whole population than ASM (Wilcoxon signed-rank test, p<0.001) and similar accuracy to TbM (Wilcoxon signed-rank test, not significant).

Template

Fig. 2 shows an example of cord straightening for images from an ALS patient. Visual evaluation revealed an asymptomatic syrinx in the MR images of one healthy subject, who was subsequently excluded from template construction, leading to a final group of 59 healthy volunteers (mean age ± SD: 44.6 ± 16.2 years, range: 21-72 years, 31 females). During the template construction step, the experienced operator had to adjust visually poor segmentations in rare occasions (error rate = 2.57%, defined as: 100 × (the number of mis-segmented axial slices)/(the total number of segmented axial slices)) using TbM [18]. Finally, images from all subjects were centered and straightened. Template and probability tissue map of the cervical spinal cord for the 59 healthy subjects are shown in Fig. 3.

Discussion

DTbM accuracy was evaluated visually by a neuroradiologist at the cervical and thoracic spinal cord levels and compared with ASM. Besides, DTbM accuracy was visually and quantitatively evaluated at the cervical and thoracic levels and compared with ASM. In addition, DTbM accuracy was quantitatively evaluated at the C2 vertebral level and compared with ASM and the well-established method for CSA measurements, namely TbM. A template and a tissue probability map of the cervical spinal cord were constructed.

Comparison of segmentation methods

DTbM showed higher accuracy for segmenting both the normal and the pathological spinal cord than ASM for the cervical and thoracic spinal cord levels. The lower accuracy of ASM can be explained by the fact that this method relies on hypotheses that are not fulfilled in the pathological spinal cord. These hypotheses are that (a) the cross-sectional shape of the cord varies only slowly in the superior-inferior direction and (b) in cross-sections, the radius from the estimated center to the edges varies smoothly around the edge [14]. Such hypotheses are valid when considering short regions of the spinal cord, for instance C2/C5 vertebral levels where the method has been validated and used to quantify atrophy in MS patients [14]. However, when considering large regions of the spinal cord (Table 1), the first hypothesis is no more valid even in healthy subjects as shown in post-mortem studies [48,49] and recently in vivo [50]. In neurodegenerative diseases, ALS is characterized by cell loss in the anterior horn and corticospinal tract degeneration [22]. Corticospinal tract degeneration induces atrophy in the lateral direction and loss of alpha motor neurons results in atrophy in the ventral direction. SMA is characterized by degeneration of cell bodies in the anterior horn [21], which potentially induces a pronounced ventral atrophy. In SCI patients, more pronounced atrophy was reported in the antero-posterior direction than in the left/right direction at C2 vertebral level [11]. Furthermore, trauma induced large spinal cord shape deformation at lesion level and around it. Therefore the second hypothesis may not be valid in ALS, SMA and SCI patients and this could explain the lack of ASM accuracy in segmenting pathological populations. As DTbM depends only on image contrast, it has the advantage of being less sensitive to variations in spinal cord shape as compared with ASM. Quantitative evaluation of DTbM and ASM compared with manual segmentation was not possible for volumetric approach in this study due to the large number of slices to be segmented. For this reason, we resorted to a visual evaluation strategy. In one control subject, visual evaluation revealed a central and focal T 2 -hypersignal at C5 vertebral level corresponding to an asymptomatic syrinx. This subject was therefore excluded from template construction. Furthermore, accuracy results of the visual evaluation were confirmed by the quantitative evaluation of CSA measurement by using DTbM and ASM at the middle of each available spinal cord vertebral level (Table 1, Table 4). The lower accuracy at the thoracic level than at the cervical level could be explained by the lower signal-to-noise ratio inherent to the thoracic coil compared with the cervical coil as well as the choice made for shimming box positioning to focus on cervical and upper thoracic levels [2,3,5,10].

A quantitative evaluation at C2 vertebral level in healthy and pathological subjects was also achieved (Table 5). Even at C2 vertebral level, our analysis showed a higher accuracy of DTbM compared with ASM and similar accuracy for DTbM and TbM. ASM showed improved accuracy than previously reported in [14]. After data preprocessing, DTbM was fully automatic and hence operator independent, which is not the case for TbM and ASM [14,18,30,51].

In regions where CSF spaces were reduced (i.e. regions of narrow spinal canal), Otsu's global thresholding method failed to extract the CSF correctly. Thus, global contrast enhancement was needed. However, this strategy did not enable initializing contours optimally with TbM leading to misestimating the spinal cord region [30]. In case this strategy failed, correction step S7 and reduction in nerve roots contribution and in the small T 2 -hyperintense region in the final segmentation result were useful (1 control, 1 ALS, 4 SCI patients). However, segmentation and correction step S7 failed in regions of large areas of T 2 -hyperintensity in the spinal cord (1 SMA patient), large regions of spinal canal narrowing with limited CSF space (1 SMA patient) as well as at the lesion level in 4 SCI patients with T 2 -hypersignal and atrophy of the spinal cord and narrow spinal canal.

Template construction

DTbM was used to construct a spinal cord template. Despite the high spatial resolution of imaging data, we were not able to determine the precise position of the nerve roots in all subjects, which would ideally enable to standardize all subjects spinal level by spinal level. Alternatively, two ways of standardization can be considered: 1) standardization to the whole median cord length [25]; 2) standardization to the median vertebral level for each vertebral level. As demonstrated previously for the cervical spinal cord [52], nerve root position showed less variation than vertebral body position. Therefore, the first strategy was expected to better preserve nerve roots position than the second. Furthermore, this strategy enabled to remove the effect of cord length in the standardized images [25]. Compared with previous studies that used ASM to construct low-resolution 1.5T MRI cervical spinal cord templates [25,27,28], the segmentation method proposed here using high-resolution 3T MR images showed improved accuracy. In addition, the new method resulted in a centered and straight template and probability tissue map.

In a recent study, a spinal cord template from 3T T2-weighted MR images and a small sample of 16 subjects was proposed with minimal manual intervention [26]. The template included the cervical and mid-thoracic regions (i. e., from C2 to T6 vertebral levels). MR images were segmented using a graph-cut segmentation method that is known to lack validation [53]. The standardization procedure consisted of a nonlinear registration of each subject's spinal canal into an average space as it is usually done when spatially normalizing brain MR images [54][55][56]. Given the small size of the spinal cord compared with CSF as well as the high inter-subject CSA variability for CSF in comparison to the spinal cord [57], nonlinear registration of the whole spinal canal is expected to increase bias when registering subjects into an average space, while in the present study we focussed on standardizing the spinal cord only. Unlike what we proposed, the standardization procedure proposed in [26] did not take inter-individual nerve roots position variability into account [58]. Besides, the error in CSA area estimation using nonlinear registration (mean error = 16%) would make the method too inaccurate for atrophy quantification in the pathological context [2,3,5,10].

Atrophy quantification in neurodegenerative diseases and trauma

Grey and white matter could not be differentiated in the acquired images due to insufficient contrast inside the spinal cord. However, as mentioned before, atrophy may predominate in a particular direction in neurodegenerative diseases and trauma. This preferential direction of cord atrophy may be detected in group analysis using surface-based morphometry techniques on spinal cord masks normalized to the template space instead of using voxel-based morphometry. For instance, morphological changes have been successfully detected in the hippocampus and ventricles in Alzheimer's disease using radial distance approach and tensor-based morphometry [59,60] and in the lateral ventricles in HIV/AIDS patients using multivariate tensorbased morphometry [61], as well as in the basal ganglia in ALS patients using surface-based vertex approach [62]. Our segmentation method may also measure accurately CSA and spinal cord volumes in a large range of pathologies.

Limitations and future directions

Manual outlining was taken as the ground truth for methods evaluation, however the accuracy of this procedure strongly depends on the operator's experience degree [63]. Nevertheless, we believe that segmentations performed by the trained operator (four years' experience) are close to the ground truth and therefore bias should be minimal. Future studies could use majority voting or simultaneous truth and performance level estimation methods to produce ground truth segmentation for method evaluation [64,65].

We did not have the opportunity to conduct a scan-rescan evaluation of DTbM. However, DTbM is based on the well-established method TbM [18], which showed low scan-rescan variability [29]. Thus, DTbM is expected to have the same order of variability as TbM, but this is worth further investigating.

Future studies could also integrate two anatomical imaging acquisitions for accurate nerve root localization [66] and for white and grey matter delineation using high-resolution T 2 Ãweighted 2D gradient recalled echo sequence, a multi-echo gradient-echo sequence (MGE) or a gradient echo with a multi-shot echo-planar imaging sequence (GE-MS EPI) [67,57]. For instance, MGE sequence has already enabled construction of a probabilistic cervical and thoracic spinal cord atlas of 15 subjects [57]. However, because of acquisition time constraint, only one slice per vertebral level was acquired (0.5×0.5 mm 2 in-plane resolution and 5 mm slice thickness). Furthermore, manual segmentation was used for grey and white matter delineation, a procedure that is time consuming and the accuracy of which strongly depends on the operator's experience degree [30,63]. GE-MS EPI sequence also enabled the construction of an atlas of the cervical spinal cord. However, multiple manual outlining was needed to construct a "gold standard" segmentation, which is extremely time consuming [68], despite the benefit in accuracy. As T 2 Ã -weighted images present a spinal cord/CSF contrast similar to that of T 2weighted images, DTbM would likely be able to segment the spinal cord accurately using T 2 Ãweighted data. Grey matter could be extracted from such images using an atlas-based segmentation approach via the template from Taso et al. (2014). Resulted masks from T2-weighted and T2 Ã -weighted images could be fused to construct a probabilistic spinal cord atlas of white and grey matter as recently proposed in [26].

Besides, as DTbM segments each axial slice independently, parallelization of segmentations could be envisaged [69], which would drastically reduce time computation (few milliseconds per slice).

Conclusion

We developed a fast and accurate semi-automated segmentation method of the spinal cord at both cervical and thoracic levels from 3T T2-weighted MR images, applied to healthy subjects and patients with various spinal cord diseases. DTbM showed higher accuracy than ASM and similar accuracy to TbM, however it clearly facilitated the whole processing by limiting the manual intervention. This enabled the construction of a cervical spinal cord template and tissue probability map. studies showed evidence of sensory abnormalities that increase with disease severity in ALS patients (Constantinovici, 1993;Gregory et al., 1993;Cosi et al., 1984). Therefore, the question of whether these alterations are consecutive to motoneurons loss, or could act as a primary debilitating mechanism of the disease remains an open question.

Supporting Information

In a previous study of ALS patients (Cohen-Adad et al., 2013), by using DTI and MT imaging, we showed that the dorsal column, i.e. pathways conveying peripheral sensory information to supraspinal structures, were altered. Diffusion tensor imaging also revealed dorsal column abnormalities in a subset of 12 patients with early onset (median/maximum disease duration 9/12.6 months). This latter, and unexpected, result suggests that the sensory afferent pathways to spinal and cortical structures might already be affected at an early stage in ALS, and thus that the sensory alteration may indeed participate in ALS pathogenesis. However, MRI does not allow a functional exploration of the pathways, spinal and/or cortical, fed by sensory afferents. Therefore, it is now necessary to verify if the anatomical changes observed with MRI in ALS patients are correlated to a dysfunction of sensory feedback integration.

The objective of the present study was to investigate the anatomo-functional properties of sensory pathways in ALS patients with distal motor dysfunctions in upper limbs and no sensory symptoms, combining spinal DTI and SEP.

Material and Methods

Twenty-one ALS patients with no clinical sensory signs and 21 age-matched controls with no history of neurological disease were recruited. The disease duration from the onset of weakness in ALS patients was 26.6±3.6 months. The spinal cord was imaged using 3T cardiac-gated high-resolution diffusion tensor imaging (DTI) (C5-T1 vertebral level, voxel size = 1×1×5 mm 3 , acquisition time ~10 min). Manual ROIs were drawn in the spinal cord to isolate the dorsal column, containing sensory afferents. Sensory evoked potentials were recorded after median and ulnar nerve stimulations.

Results synthesis

We coupled spinal DTI and electrophysiological recordings for the first time to evaluate the anatomo-functional properties of sensory pathways in humans. This combined approach revealed subclinical sensory defects in 85% of ALS patients, while separated methods revealed abnormal values in 60% (Figure 5). Outcome measures: Fractional anisotropy and diffusivity of the dorsal columns at C5-T1 levels (DTI metrics) and SEPs after median and ulnar nerve stimulations (latency and amplitude of N9 and N20 components).

Results: Abnormal DTI metrics indicated anatomical damages of ascending sensory fibres in ∼60% of patients (p<0.05). Raw SEPs (μV) were smaller in ∼40% of patients but the difference with healthy subjects was not significant ( p>0.16). Their normalisation to prestimulus activity strengthened the difference between groups ( p<0.05) and allowed identification of ∼60% of patients with abnormal values. According to N9 latency, the peripheral conduction time was normal in patients ( p>0.32) but based on N20 latency, the central conduction time (between spinal cord and parietal cortex) was found to be slower (p<0.05). Significant correlation was found between DTI metrics and N9 amplitude (p<0.05). Altered SEPs were also correlated with the disease duration (p<0.05).

Taken together, spinal imaging and electrophysiology helped to identify ∼85% of patients with subclinical sensory defect while separated methods revealed abnormal values in ∼60%.

Conclusions: Sensory impairments have been underestimated at early stages of ALS. These results show for the first time the interest to combine electrophysiology and imaging to assess non-motor system involvement in ALS.

Trial registration number: IDRCB2012-A00016-37.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease characterised by degeneration of upper as well as lower motor neurons. Postmortem anatomopathology has also revealed degeneration of sensory fibres but to a lesser extent than motor axons, and it has been suggested that the sensory system might be similarly affected as the motor system, but the degeneration is less advanced at the time of death. 1-3 Peripheral and central degeneration of sensory pathways have also been reported in presymptomatic ALS mice, suggesting that the sensory defect occurs at a very early stage of the degenerative process. 4-6 Accordingly, sensory nerve biopsy 7 and multiparametric spinal imaging of the dorsal column 8 1 involvement in the degenerative process in ALS is still unknown. From a clinical perspective, while discreet sensory clinical impairment does not exclude ALS diagnosis, it is declared atypical or even excluded when sensory abnormalities are major. 9 10 Because of these uncertainties, better characterisation of the sensory involvement in well-characterised populations of ALS is needed.

Sensory nerve action potentials (SNAPs) and somatosensory evoked potentials (SEPs), routinely used to assess sensory pathways, have been investigated extensively in patients with ALS. Except in three studies, 11-13 abnormal SEPs were reported, on average, in about 1/3 patients without clinical sensory deficits. 7 14-22 The abnormality increases with disease severity, 20 23-28 and is more common when stimulating lower limb peripheral nerves than those of upper limbs. 16 17 25 29 However, the anatomical substrate of altered SEPs demonstrated in previous studies remains disputed. Indeed, altered cortical SEPs were not always associated with abnormal SNAPs and/or cervicobrachial SEPs (N9). Moreover, altered SNAPs were most often observed in patients exhibiting sensory signs or symptoms, or were sometimes related to associated medical conditions ( polyneuropathy, median or ulnar nerve entrapment neuropathies). 12 20 25 27-30 In line with this, the various components of cortical SEPs are not altered unequally, whether in amplitude and/or latency, and given the multiple origins of the SEP components, 31 some authors have suggested that abnormal SEPs in ALS are not due to sensory defects but to abnormal corticocortical interactions. 24 32 Until now, it has therefore been difficult to establish a link between anatomical and functional alterations of sensory pathways in ALS, given that the latter are based on SEPs, that depends on peripheral afferent inputs and cortico-subcortical excitability. 31 Moreover, the origin of the neural sources, discrimination of which is based on the latency of SEP components, becomes uncertain when SEPs are delayed. Thus, the aim of the present work was to test, in typical patients with ALS with distal motor dysfunctions in upper limbs and no sensory symptoms, whether abnormal metrics of dorsal column imaging are associated with altered SEPs, with a focus on N9 and N20 components, which depend mainly on the peripheral afferent volley. 31 In addition, to control for experimentdependent variability of electrophysiological measurements, a limitation for the interpretation of previous studies, we propose to normalise SEP amplitude to improve evaluation of interindividual differences, as performed when studying muscular recordings. 33

METHODS

Participants

Inclusion criteria were: (1)

Spinal diffusion tensor imaging

Image acquisitions were performed using a 3 T MRI system (TIM Trio, Siemens Healthcare, Erlangen, Germany) and neck/spine coil. Cardiac-gated diffusion tensor imaging (DTI) was performed using a single shot echo-planar imaging (EPI) sequence with monopolar scheme. The axial slice was positioned perpendicular to the spinal cord, in the middle of each vertebral level between C2-T2 vertebral levels (8 slices) but the acquisition was optimised for C5-T1 (figure 1A). Imaging parameters were: field of view =128 mm, TR/TE=700/96 ms, voxel size =1×1×5 mm 3 , parallel acquisition: R=2, b-value 1000 s/mm 2 , 64 directions, 2 repetitions, acquisition time 10 min. For a comprehensive description of the MRI acquisition parameters, the readers are invited to refer to a previous paper. 34 Electrophysiology Electrophysiological investigations were performed the same day as the MRI in each participant. Median and ulnar nerves were stimulated (1 ms rectangular electrical stimulation; DS7A, Digitimer Ltd, Hertfordshire, UK) at the wrist level using bipolar surface electrodes (0.5 cm 2 , 1 cm apart). All controls were stimulated on the dominant side, according to The Edinburgh handedness inventory; 35

Data analysis DTI

Motion correction was applied slice-by-slice using FSL FLIRT with three degrees of freedom (Tx, Ty, Rz). Diffusion tensor and its related metrics, fractional anisotropy (FA), axial (λ ⁄⁄ ) and radial (λ ⊥ ) and mean diffusivity (MD), were estimated on a voxel-wise basis. The region of interest (ROI), limited to the hemidorsal region ipsilateral to the electrical stimulations, was manually defined on each slice using geometry-based information. To avoid any user bias, the ROI was defined on the mean diffusion weighted images by an experienced segmentation operator (MMEM; figure 1A). 8 34 Furthermore, during ROIs classification, the operator opted for a conservative approach by excluding pixels that were at the cord/corticospinal fluid or white matter/grey matter interfaces.

Electrophysiology

Peak latencies of N9 (Erb's point) and N20 ( parietal) were evaluated in each subject. The peripheral conduction velocity (CVp) was calculated according to the distance between stimulating electrodes and C7 vertebra and N9 latency. The central conduction velocity (CVc) was calculated according to the distance between C7 and parietal cortex and the difference between N9 and N20 latencies. Peak-to-peak amplitude of N9 (figure 3A,B) and amplitude of the first positivity of N20 (figure 3C,D) were measured and normalised to the prestimulus activity (calculated over a 100 ms period, excluding the stimulus artefact). Given the variability of prestimulus activity, the SD of prestimulus activity was calculated and multiplied by 2 (2×SD) to define the positive and negative limits of background activity (dotted lines in figure 3A,D). This procedure is commonly used to determine when the activity significantly changes from background activity after stimuli. Because these limits give a good estimation of prestimulus activity amplitude, we used the procedure to normalise the amplitude of SEPs.

Statistics

First, each DTI and electrophysiological metric was independently compared between groups using unpaired t tests. Then, the metrics found to be significantly modified in patients were retained for further analyses to test the relationships between these metrics and clinical features for the group of participants. Thus, multiple regression analyses were performed to test the link between λ ⊥ , MD, N9, N20 and CVc according to the subject groups.

In addition, Pearson correlation analyses were performed to investigate the relationships between clinical features and λ ⊥ , MD, N9 and N20. The 95% CI of control data was calculated for DTI and electrophysiological metrics. These intervals were used to fix the limits of normal values in order to identify the patients exhibiting abnormal data with a risk of error less than 5%. Statistical analysis was conducted using StatEL software (http://www. adscience.eu) and the significance level was set at p value <0.05. Arithmetical means are indicated ±1SEM.

RESULTS

Spinal DTI metrics

The box plot charts in figure 1B, C show the distribution of λ ⊥ and MD, respectively, in the hemidorsal region of the spinal cord, corresponding to the posterior column-medial lemniscus pathway ipsilateral to electrical stimuli delivered when investigating SEPs (see below), in 20 controls and 19 patients (due to artefacts, 1 control and 2 patients were excluded). On average, both metrics were significantly higher in patients than in controls (unpaired t test, p<0.05). Abnormal values of λ ⊥ and MD were observed in 11/19 patients (57.9%). The differences in λ ⊥ and MD were not accompanied by a significant change in FA or λ ⁄⁄ ( p=0.29 and 0.06, respectively). Similar analyses were also performed on the lateral region corresponding to the lateral corticospinal tract. On average, λ ⊥ and MD were also higher in patients than in controls ( p<0.01 and 0.05, respectively) and FA was smaller ( p<0.05); λ ⁄⁄ was not significantly different between groups ( p=0.42).

Electrophysiological measurements

Figure 2 shows the mean PT and MT for median and ulnar nerves in controls and patients. While PT for both nerves was similar between controls and patients ( p=0.93 and 0.73 for median and ulnar nerves, respectively; figure 2A), MT was higher in patients, reaching the statistically significant level for the ulnar nerve ( p<0.05 and 0.12 for the median nerve; figure 2B). The stimulus intensity used for evoking SEPs (9×PT) was above MT for both nerves in all controls and in 16/21 patients for the median nerve and in 18/21 patients for the ulnar nerve.

Significant difference between groups was not observed in peak latency for N9 (for which one patient was excluded because of artefacts) nor for N20 (table 2; 0.25<p<0.93). N9 latency only depended on the peripheral conduction time (between stimulating electrodes and Erb's point) and was thus used to estimate the CVp. For both nerves, the CVp was found to be similar in patients and controls ( p=0.56 and 0.32, in median and ulnar nerve, respectively). The CVc (between spinal cord and parietal cortex) was estimated by subtracting N20 and N9 latencies. It was slower in patients than in controls, reaching statistical significance for the median nerve only ( p<0.05, and 0.08 for the ulnar nerve).

Figure 3A, D shows the raw signals in one control (AC) and 1 patient (BD). Both N9 (AB) and N20 (CD) were smaller in the patient compared to the control. On average, the raw amplitude (in μV) of both N9 and N20 was smaller in patients than in controls, but the difference did not reach statistical significance (0.16<p<0.42; table 2). When normalised, the difference between groups became significant ( p<0.01 and 0.001 for N9 in median and ulnar nerve, respectively, and p<0.05 for N20; figure 3E,F), which confirmed that the peripheral volley (N9) and the first component of early cortical SEP (N20) were smaller in the patient group. No significant correlation was observed between the amplitudes of N9 and N20 (Pearson, p=0.67).

Abnormal N9 was observed in 17/20 patients (85%) for the ulnar nerve and in 9/20 patients (45%) for the median nerve. Abnormal N20 was observed in 11/21 patients (52.4%) for the ulnar nerve and in 10/21 patients (47.6%) for the median nerve. On average, the SEP normalisation helped to identify 57.5% of patients with abnormal values while 41.3% exhibited abnormal raw data (in μV).

Relationships between DTI and electrophysiological metrics

The scatter plots in figure 4 show the dispersion of N9 and N20 amplitudes according to MD (dorsal column ipsilateral to stimulation site) for controls and patients, for median as well as ulnar nerves (Dixon test did not detect any aberrant values). Multiple regression analyses further confirmed the significant difference between groups, in N9 and N20 amplitudes. In addition, significant relationships were found between DTI metrics and N9 (except for λ ⊥ and N9 from ulnar) but not with N20 (see statistics in table 3). Similarly, significant relationships were observed between the CVc and λ ⊥ ( p<0.05) and MD ( p<0.01) for the median nerve.

Figure 5 shows that a larger proportion of patients exhibited abnormal DTI metrics and/or evoked potentials (84.7±3.8% on average; red portion) compared to patients having normal metrics (white portion). Among the patients exhibiting at least one abnormal metric, 44.4±7.1% have both abnormal DTI and evoked potentials ( purple subportions). Similar proportions were found when comparing the DTI metrics and the CVc.

Correlation with clinical features

Multiple regression analyses were performed to investigate the relationships between DTI and electrophysiological metrics and clinical features. λ ⊥ , MD, N9 and N20 were not correlated with any clinical features related to hand motor performance (ALSFRS-R subscore for handwriting, hand muscle testing, nine hole peg test; 0.27<p<0.79).

Pearson correlation analyses revealed a significant depression of N20 according to the disease duration (R 2 =0.17 and 0.15 for ulnar and median nerve, respectively; p<0.05). N9 significantly decreased with disease duration for median nerve only (R 2 =0.19, p<0.05; R 2 =-0.05, p=0.74 for ulnar nerve). No significant relationship was found between disease duration and spinal DTI metrics (p>0.3).

DISCUSSION

The main result of the present study is that by coupling spinal DTI and electrophysiology, almost 85% patients with ALS with subclinical sensory defect in an upper limb could be identified. Considered separately, each technique revealed abnormal values in less than 60% patients. The second novelty of this study is that normalisation helped to identify more patients with abnormal SEP from a 40% detection rate when expressed in μV, to 60% when taking into account the level of prestimulus activity. This study has also shown a significant relationship between DTI metrics and N9, and a significant depression of evoked potentials with disease duration.

Reliability of spinal DTI

Spinal DTI metrics were found to be significantly altered in patients, which gives further support for early anatomical damage of the dorsal columns in ALS. Compared to our previous study, 8 the patients in the present study exhibited less motor weakness especially in proximal upper limb muscles. Accordingly, λ ⊥ for the lateral corticospinal tracts was lower in the present study compared to the previous one, which supports a less severe impairment of the motor pathways in the patients investigated here. Moreover, while the relationship between motor and sensory deficits has not been characterised so far, some studies have reported increased SEP alteration with the disease severity. 20 23-28 Given that the degeneration is likely to be slower in sensory pathways than in motor pathways, 2 one would expect a smaller λ ⊥ for the dorsal columns in the present study, compared to the previous one, 8 which was the case. Thus, the comparison between the two studies indicates that spinal DTI is a reliable and very sensitive method for detecting anatomical tract defects at spinal level. 36 Some studies on visual evoked potentials in humans have revealed the influence of the level of prestimulus EEG activity on the size of the evoked potentials and reported better reliability with normalised data. 37 38 Here, SEPs were normalised to 2×SD of prestimulus activity, which is commonly used to evaluate the level of activity before conditioning, and to determine when the poststimulus signal deviates significantly from background activity. Normalisation of evoked potentials to this value allowed the basal activity to be taken into account and reduced the variability due to noise. Comparison of normalised data strengthened the differences between groups, helped to identify more patients with abnormal values and indicated a greater mean suppression from 20% (in raw data) to 50% (when normalised) for N9 and <5% to 20% for N20 (according to the mean values in controls). The comparison of raw and normalised data suggests that abnormal SNAPs and SEPs in ALS might have been previously underestimated.

Alteration of peripheral volleys

Histological evidence for sensory nerve degeneration has only been found in lower limbs with sural nerve biopsies. 3 In upper limbs, abnormal SNAPs were found mainly in patients with clinical sensory signs. 7 12 20 25 27 29 30 To some extent, we found analogous results because the characteristics of N9 (Erb's Latency: mean peak latency (ms±1 SEM) of N9 recorded at the Erb point, and of N20 recorded at the level of primary somatosensory cortex (S1) for median and ulnar nerves, in controls and patients with amyotrophic lateral sclerosis. CVp: peripheral conduction velocity (m/s±1 SEM) according to N9 peak latency and the distance between stimulation site and C7 vertebra. CVc: central conduction velocity (m/s±1 SEM) according to the difference between N20 and N9 peak latencies and the distance between C7 vertebra and recording site, in front of S1. Amplitude: peak-to-peak amplitude of N9 and peak amplitude of N20 (μV). *p<0.05. Open Access point) were as in controls: similar peak latency and similar CVp with no significant change in the mean raw amplitude (in μV). However, abnormal raw values were observed in 20-50% of patients and the normalisation strengthened the difference between groups, which further supports subclinical peripheral abnormalities. 27 Our group of patients was very homogenous in regard to hand muscle weakness in the median and ulnar nerve territories, which might account for the higher proportion of patients exhibiting abnormal N9 compared to previous studies. 21-23 Alternatively, a reduced antidromic volley in motor axons may account for N9 depression. However, a study performed in patients with pronounced or complete loss of motor axons in the median nerve failed to reveal any change in N9. 39 According to hand motor weakness, we found a higher MT in patients and, in some, the conditioning stimuli used to evoke SEPs were below MT. However, these patients did not exhibit the smallest N9 and there was no link between abnormal N9 values and MT. Lastly, the relationship between spinal DTI and N9 further supports the fact that the latter depends mainly on the peripheral sensory afferent volley 31 and its alteration in ALS.

Origin of the impairments along the sensory pathways

The present changes in spinal DTI metrics give further evidence for anatomical impairments of sensory pathways in ALS. The alteration of N9 amplitude, without changes in peripheral conduction velocity, suggests loss of peripheral sensory axons. 3-6 The first cortical component N20 was delayed and depressed to a lesser extent than N9. The absence of correlation between the alterations of N9 and N20 can be explained by the fact that at a stimulus intensity producing maximal N20, N9 reached half its maximal size, and this intensity can be at or slightly above MT. 40 Therefore, at the stimulus intensity we used (9×PT, which was above MT in most participants), N20 was likely to be maximal and, because of saturation, the measurements might have been less sensitive at a cortical level than at a peripheral level. However, N20 not only depends on the sensory afferent inputs, but also to central processing. 40 Accordingly, a recent cerebral MRI study has shown atrophy in the thalamus, 41 suggesting that the thalamocortical relay for sensory information may be altered in ALS. In line with this, we found a slower CVc in patients than in controls, without any change in CVp. Therefore, abnormal N9 and N20, together with impaired spinal DTI reported here, give strong evidence for subclinical sensory defects in ALS at peripheral as well as central levels.

Comparing median nerve and skin stimuli suggests that N20 mainly depends on cutaneous inputs to the 3b area. 31 42-44 Thus, altered N20 from median nerve stimulation could suggest impaired cutaneous fibres. However, if low-threshold cutaneous afferents were affected, this would have influenced PT, which was found unchanged. Alternatively, changes could be due to proprioceptive afferents. Cutaneous fields innervated by median nerve are larger than those innervated by ulnar nerve but 75% intrinsic hand muscles are innervated by ulnar nerve. Additionally, SEPs from mixed nerves in lower limbs, probably more motor than the median nerve (such as ulnar nerve), are mainly produced by proprioceptive afferents. 45 Therefore, impaired proprioceptive inputs would affect SEPs produced by ulnar nerve more than those produced by median nerve, which was the case.

The absence of clinical sensory signs, of correlation between sensory defect and motor symptoms and the fact that DTI metrics for the lateral corticospinal tract were more affected than those for the dorsal columns suggest that the impairment of sensory pathways was less than that of motor systems. However, the correlation with disease duration suggests that the degeneration of sensory pathways is progressive.

CONCLUSIONS

The combination of normalised electrophysiological measurements and spinal DTI metrics suggest that sensory deficits in ALS have been previously underestimated and gives further evidence for early degeneration in sensory pathways at peripheral as well as central levels. These results show, for the first time, the benefit of combining electrophysiology and biomedical imaging to assess non-motor system involvement in ALS.

Study 4 -Multi-parametric spinal cord MRI as potential progression marker in amyotrophic lateral sclerosis

Background and objectives

Recent works by our team and others showed that multi-parametric MRI approaches (DTI, MT and atrophy measurement) allow quantification of lesions in ALS (Cohen-Adad et al., 2013;Agosta et al., 2009;Valsasina et al., 2007) and detection of infraclinical lesions (Study 3). In addition, we showed that some metrics correlated with LMN involvement, which is reflected by cord atrophy, as assessed by transcranial magnetic stimulation (TMS) results (Cohen Adad et al., 2013). Importantly, we showed a correlation between cord atrophy in ALS patients and clinical disability (Cohen-Adad et al., 2013), that was later confirmed in an independent cohort (Branco et al., 2014). In the context of ALS, which among the neurodegenerative diseases is characterized by its fast progression, investigating the progression of lesions is a major challenge. From a pathophysiological perspective, there is a growing interest in the mechanisms of propagation of ALS with the emerging hypothesis of prion-like propagation [START_REF] Pradat | Deciphering spreading mechanisms in amyotrophic lateral sclerosis: clinical evidence and potential molecular processes[END_REF]. Neuroimaging methods appear as necessary tools to study in-vivo the temporal and spatial pattern of propagation [START_REF] Pradat | Deciphering spreading mechanisms in amyotrophic lateral sclerosis: clinical evidence and potential molecular processes[END_REF]. Longitudinal studies are also mandatory to test and validate biomarkers of progression that are an unmet need for monitoring disease progression at the individual level and to provide surrogate markers of efficacy in clinical trials (Annex A). The development of high motor disability and respiratory insufficiency with orthopnea are classical limitations for performing longitudinal studies in ALS patients.

The main objective of the present study was to test the hypothesis that spinal cord MRI was able to detect in-vivo the progression of the neurodegenerative lesions in ALS. The second objective was to test whether the changes of MRI metrics were clinically meaningful, that is, a major criteria for biomarker development, by investing correlations with functional decline.

Material and Methods

After a first MRI (Cohen-Adad et al., 2013), 29 ALS patients were clinically followed during 12 months; 14/29 patients underwent a second MRI at 11±3 months. Cross-sectional area that has been shown to be a marker of LMN degeneration was measured in cervical and upper thoracic spinal cord from 3D T2-weighted images (Cohen-Adad et al., 2013). Diffusion tensor imaging and MT imaging metrics were measured within the lateral CST in the cervical region (from C2 to C7). Patients were clinically assessed and scored on the ALSFRS-R and manual muscle testing (MMT) score.

Results synthesis

Our longitudinal study showed that it was possible to detect a change of MRI metrics over time.

We were able to detect a significant decrease in the lateral segment of the spinal of MTR by 0.33 units/month and by 0.14 mm²/month for cross-sectional area. In the context of ALS, it outlines the accuracy of our multi-parametric MRI approach in detecting the progression of neurodegeneration in ALS. Interestingly, we were not able to detect a significant change over time in DTI metrics that reflected UMN degeneration.

Our results suggest that the cross-sectional area is the most reliable marker of longitudinal neurodegeneration in the spinal cord compared to MMT or DTI metrics such as fractional anisotropy or radial diffusivity. Our study also demonstrated a strong relationship between the spinal cord atrophy rate of change and clinical progression (ALFRS-R and MMT). These results are promising for development of biomarkers in ALS. Here again, further longitudinal studies in a larger population are needed to validate spinal cord atrophy as a surrogate marker of disease progression. An important aspect is to evaluate the sensitivity of multi-parametric MRI metrics to change over time, i.e. whether MRI is able to detect significant changes earlier than classical clinical scores, e. g. ALSFRS-R and MMT.

Introduction

Amyotrophic Lateral Sclerosis (ALS) is the most frequent motor neuron disease characterized by degeneration of both upper and lower motor neurons. Disease progression is characterized by worsening of weakness and physical disability with a median survival ranging from 2.5 to 3 years [1,2]. There is no curative treatment and the only available neuroprotective drug is riluzole that showed only a modest effect on survival but no evidence of effect on the progression of disability [3]. There is an unmet need for biomarkers in amyotrophic lateral sclerosis, not only for better diagnosis but also to assess disease progression and the effect of disease-modifying drugs in clinical trials (surrogate markers) [4,5,6]. There is an increasing body of knowledge suggesting that new neuroimaging approaches, including diffusion tensor imaging (DTI) and magnetization transfer imaging, may provide promising biomarkers in ALS [7][8][9][10].

MRI studies in ALS mostly investigated neurodegenerative changes in the brain and thus only investigated upper motor neuron involvement [11][12][13][14][15]. Spinal cord MRI has the advantage of investigating the two motor system components that are involved in ALS, i.e the lower motor neuron (via gray matter atrophy) and upper motor neuron (via degeneration of corticospinal tract). However, spinal cord imaging has technical limitations due to the small diameter of the spinal cord, physiological motions (respiratory and cardiac movements) and susceptibility artifacts [16,17,18]. Recently, progress in neuroimaging protocols using multi-parametric MRI approaches (DTI, MT and atrophy measurement) allowed to detect abnormalities in the cervical cord of ALS patients [10,[19][20][21][22], some metrics being correlated with upper or lower motor neuron involvement as assessed by transcranial magnetic stimulation (TMS) results [10]. Two transversal MRI studies showed a correlation between CSA and clinical disability [10,22]. For MRI metrics to be used as surrogate marker of neurodegeneration, longitudinal studies are mandatory to determine whether these metrics are sensitive to changes over time and correlate with clinical progression. To address this issue we performed a spinal cord MRI longitudinal study in a series of ALS patients.

Materials and Methods

Subjects

Twenty-nine patients with probable or definite ALS were enrolled in the study and underwent a baseline spinal MRI examination (MRI 1 ). A follow-up MRI (MRI 2 ) was performed in 14 of these patients after a mean delay of 11.1 months (62.7 months). Follow-up MRI was not feasible in other patients due to orthopnea (n = 9), worsening of the general condition rendering transportation and MRI examination impossible (n = 1), loss of follow-up (n = 2), death (n = 2) and refusal (n = 1). The local Ethics Committee of our institution approved all experimental procedures (Paris-Ile de France Ethical Committee under the 2009-A00291-56 registration number), and written informed consent was obtained from each participant. All clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki.

Revised ALS Functional Rating Scale [23] data were obtained at MRI 1 , one year after MRI 1 in all 29 ALS patients and at MRI 2 for the 14 ALS patients. We calculated an arm ALSFRS-R subscore pertaining to functions such as handwriting, cutting food and handling utensils, and a leg ALSFRS-R subscore (walking, climbing stairs). The patients were also scored on manual muscle testing (MMT) using the Medical Research Council score [24]. MMT was performed at baseline in all ALS patients and at one year in the subgroup of patients who had a second MRI. Seven muscles in each limb were assessed. Information about patients' demographics and clinical disability are given in Table 1. It includes disease duration, which was defined as the delay between the onset of weakness and MRI examination. Patients were scored on the day of each MRI.

MRI Acquisition

Acquisitions were performed using a 3T MRI system (TIM Trio, Siemens Healthcare, Erlangen, Germany), with a body coil for signal excitation and a neck/spine coil for signal reception. Anatomical imaging was performed at the cervical and upper thoracic levels. DTI and magnetization transfer imaging were performed from vertebral C2 to T2 using the following protocol:

Anatomical data. A sagittal T2-weighted three-dimensional (3D) turbo spin echo (TSE) image with slab selective excitation was acquired. Imaging parameters were: isotropic voxel size 0.9 mm 3 ; FOV = 2806280 mm 2 ; 52 sagittal slices; TR = 1500 ms; TE = 120 ms; acceleration factor = 3; acquisition time , 6 min.

Diffusion weighted-imaging. DTI data were acquired using a single shot EPI sequence with monopolar diffusionweighting scheme. The acquisition was cardiac-gated. Eight axial slices covering C2 to T2 vertebral levels were acquired. Imaging parameters were: voxel size = 16165 mm 3 ; FOV = 1286128 mm 2 ; TR = 700 ms; TE = 96 ms; acceleration factor = 2; bvalue = 1000 s/mm 2 ; 64 diffusion encoding directions; 4 averages; acquisition time , 15 min.

Magnetization transfer. 3D gradient echo images with slabselective excitation were acquired with and without magnetization transfer (MT) saturation pulse (Gaussian envelope, duration 9984 ms, frequency offset 1200 Hz). Imaging parameters were: voxel size = 0.960.962 mm 3 ; FOV = 2306230 mm 2 ; axial orientation with 52 slices (covering the same C2-T2 region as the DTI scans), TR = 28 ms; TE = 3.2 ms; acquisition time ,5 min per volume.

For an exhaustive description of the MRI acquisition parameters, the reader is referred to [25].

TMS Examination

TMS was performed within two weeks of the MRI 1 using a MAGSTIM 200 device, delivering monophasic stimulation through a round coil (9 cm diameter). Responses of hand muscle (adductor digiti minimi) were recorded with surface electrodes using a KPnet system (Natus/Dantec, Denmark). The stimulation was first applied at the cervical level (C7-D1) to excite the root near its exit from the spinal cord [26]. The motor evoked potential amplitude was measured to assess lower motor neurons impairment. The motor evoked potential threshold was measured to assess CST degeneration [27] (Table 1).

DATA Processing

Cord atrophy. Cord cross-sectional area (CSA) was measured by an experienced operator on the segmentation technic (i.e. four years experience) on the T2-TSE images in the middle of vertebral levels from C2 to T6 using the semi-automatic method that has been shown to be accurate and with a low inter-observer variability [28,29,30]. The plane perpendicular to the spinal cord was resampled to minimize partial volume between spinal cord and cerebrospinal fluid [31]. To increase reproducibility and accuracy of the used segmentation method, images were segmented by an experienced operator on the technic. DTI. Data were corrected for motion slice-by-slice using FSL FLIRT [32] with three degrees of freedom (Tx, Ty, Rz). Diffusion metrics were estimated voxel-wise using FSL DTIFIT: fractional anisotropy (FA), radial diffusivity (l H ), axial diffusivity (l // ) and mean diffusivity (MD).

Magnetization transfer. Gradient echo volumes with and without magnetization transfer pulse were coregistered using FSL FNIRT non-linear algorithm. Magnetization transfer ratio (MTR) was computed voxel-wise following the equation 100 6 ((S 0 2 S MT )/S 0 ), where S 0 and S MT represent the signal without and with the magnetization transfer pulse, respectively. ROI-based analysis. The lateral portion of the cord was delineated manually and using geometry-based information by an experienced operator on segmentation. To minimize bias, these regions of interest (ROIs) were defined on the mean diffusion weighted images (for DTI analysis) and on the 3D gradient-echo T1-weighted image (for MT analysis) (Figure 1). This method has been shown to be sensitive enough to discriminate changes between sensory (posterior) and motor (lateral) tracks in the context of ALS [10] and spinal cord injury [25]. For an exhaustive description of ROIs definition, the reader is referred to [25].

Statistics

Statistical analysis was performed using Matlab (The Mathworks Inc, MA, USA).

Correlations of MRI metrics with clinical disability at baseline.

Stepwise linear regression was used to find the best predictor of clinical disability at MRI 1 in all 29 patients. Dependent variables were: ALSFRS-R (total, arm subscore and leg subscore) and MMT. Predictors were: gender, age, site onset, disease duration, FA, l H , l // , MD, MTR, cross-sectional area, MEP threshold and amplitude. The probability for a predictor to enter the stepwise model was based on a Fisher's test, with a Pvalue set to 0.05.

Prediction of clinical disability after one-year followup.

Stepwise linear regression was used to find the best predictor of clinical disability in all 29 ALS patients one year after MRI 1 . Dependent variable was: ALSFRS-R (arm, leg and total scores) after one-year follow-up. Predictors were: gender, age, site onset, disease duration, FA, l H , l // , MD, MTR, cross-sectional area, MEP threshold and amplitude measured at baseline. The probability for a predictor to enter the stepwise model was based on a Fisher's test, with a P-value set to 0.05.

Longitudinal MRI Study

Results

Correlations of MRI Metrics with Clinical Disability at Baseline

Cord CSA was the only significant predictor associated with the arm subscore of ALSFRS-R (P = 0.049) and MMT (P = 0.019 for arm and 0.04 for leg) scales. FA measure in the lateral segment of the spinal cord was the only predictor of leg subscore (P = 3.10 24 ) and total score (P = 0.039) of the ALSFRS-R. Results of stepwise linear regression analysis are summarized in Table 2 (Bonferroni non corrected, significance level alpha = 0.0056).

Prediction of Clinical Disability after One-year Follow-up

Results of the stepwise linear regression analysis revealed that the mean spinal cord CSA at MRI 1 was the only predictor of the arm ALSFRS-R subscore (P = 0.013, Table 3). FA measured at MRI 1 was the only predictor of the leg ALSFRS-R subscore (P = 0.002). FA, l H and CSA measurements predicted the total ALSFSR-R score (P FA = 2.10 24 , P lH = 0.009, P CSA = 0.014). There was no predictor of the decline of the arm or leg ALSFRS-R subscores (P.0.05) (Bonferroni non corrected, significance level alpha = 0.0056). Comparisons between Baseline and Follow-up MRI MRI metrics for MRI 1 and MRI 2 in ALS patients are indicated in Table 4.

The cord CSA decreased in all patients, except in one case, by a mean of 0.14 mm 2 /month (P = 9.10 24 ). Only one patient did not show a decrease in spinal cord area (minor increase of 0.04 mm 2 / month), within the range of sensitivity of the segmentation method [30]. Furthermore, this patient had a very long disease duration (9 years) and was clinically stable between the two MRI. MTR decreased by 0.33 units/month (P = 0.003). Figure 2 shows plots of CSA and MTR at baseline and follow-up.

No significant change was detected either for FA (P = 0.168), l H (P = 0.127), l // (P = 0.052) or for MD (P = 0.068) (Bonferroni corrected, significance level alpha = 0.008).

Correlation between Changes in MRI Metrics Over Time and Progression of Clinical Disability

Results of correlations are shown in Figure 3. The atrophy rate of the spinal cord area (i.e., CSA rate of change) was highly correlated with worsening in clinical functional status, the correlations being higher when considering the arm subscore of the ALSFRS-R (R = 0.702, P = 0.005) and the arm subscore of the MMT (R = 0.717, P = 0.004). No correlations were detected between the atrophy rate of change and rates of changes in the leg subscore of the ALSFRS-R (R = 0.376, P = 0.185), the total ALSFSR-R (R = 0.481, P = 0.084), and the leg subscores of the MMT (R = 0.153, P = 0.601).

No correlations were detected between the rate of change in MTR and the rates of changes in the arm ALSFRS-R (R = 0.037, P = 0.914), leg ALSFRS-R (R = 20.432, P = 0.184), arm MMT (R = 20.059, P = 0.863) and leg MMT (R = 0.009, P = 0.979) subscores (Bonferroni corrected, significance level alpha = 0.025).

Discussion

Results show that spinal cord atrophy and MTR are sensitive the progression of the tissue neurodegenerative process in the spinal cord in ALS patients. The cord area at baseline was predictive of the functional ability of the arms after a one-year follow-up duration. Between MRI 1 and MRI 2 , MTR decreased significantly in the lateral segments of the spinal cord by 0.33 units/month and the cross sectional area decreased significantly by 0.14 mm 2 /month. Furthermore, the atrophy rate of change was strongly correlated with the rates of change in the arm ALSFRS-R and MMT subscores.

Significant decrease in cord area in ALS patients followed longitudinally was previously reported [20], although no correlation with the ALSFRS score was found. Several factors may explain this lack of correlation in the study of Agosta et al. First, the lower field strength (1.5T versus 3T) might have been associated with lower resolution and/or lower signal-to-noise ratio, yielding less precision in delineating the spinal cord. Second, the present study includes a larger rostro-caudal portion of the spinal cord, which may have increased the sensitivity to detect motor neuron degeneration affecting the ALSFRS score. Third, the follow-up period was shorter (9 versus 11 months in the present study) and patients showed a slower ALSFRS decline (0.5 versus 0.8 in the present study). Finally, variability in the population studied, given the small sample sizes, may also account for the differences. Among our cohort of 29 patients, a follow-up MRI was only feasible in 14 patients. This relatively high level of dropout rate (52%) is common in longitudinal studies in ALS (from 27% to 75%, depending on the duration of the follow-up) [20,[33][34][35][36][37][38][39]. The development of respiratory insufficiency responsible for orthopnea and severe disability are classical limitations for performing a re-scan in ALS patients.

Our result suggest that changes over time of spinal cord MRI metric may reflect to some extent the respective contribution of lower and upper motor neuron degeneration to disability. Changes of FA measured in the lateral spinal cord containing mostly the cortico-spinal tract were linked to leg disablity. These results are reminiscent of clinical observations, since symptoms due to upper motor neuron degeneration, particularly spasticity, mainly affect the lower limb. Stiffness in the lower limb, which is associated with balance and posture impairment, is a major cause of disability even in ALS patients without noticeable motor deficit in the lower limbs [40]. In our previous study, we showed that CSA was correlated with MEP amplitude, that is a TMS index of lower motor neuron involvement, and not with MEP threshold, that is a TMS index of upper motor neuron involvement [10]. So as, the observed correlations between atrophy rate of change and arm functional sub-scores decline suggest that lower motor neuron degeneration was the determinant factor of the arm disability progression. However, future study using higher resolution enabling separation between grey and white matter may provide more arguments to support these assumptions.

It is possible that cord atrophy partly contributed to the longitudinal decrease in MTR due to partial volume effects with adjacent CSF and gray matter, which may have lowered the apparent MTR calculation from averaging effects. However, several arguments support that partial volume effect minimally contributed to the detected effect. Firstly, although cord area was significantly smaller at follow-up, the variation was about 1.6 mm 2 , which corresponds to a change in radius of less than five microns (using circular approximation of the cord). Given that pixel size is 161 mm 2 , the partial volume effect resulting from the coarse gridbased definition of the ROIs-which only encompass 10-15 voxels over the corticospinal region-dominates in comparison to the change in cord diameter. In other words, the variability introduced by the coarse sampling of MRI markers is orders of magnitude higher than the systematic bias introduced by a small change in the cord size. Secondly, during delineation of the cord we opted for a conservative approach, by leaving out pixels that were at the cord/CSF or white matter/gray matter interfaces. Although this approach has flaws, this is the currently adopted approach for quantifying MRI metrics in the spinal cord. Thirdly, if it were a pure partial volume effect, differences would also be observed in the diffusion metrics submitted to the same processing (spatial resolution was almost the same for DTI and MTR). However, neither significant difference nor trends were observed for diffusion metrics between baseline and follow-up. Although previous studies introduced atrophy as a confound for isolating the effect of a dependent variable [41], it is often a delicate process due to the high correlation between atrophy and other MRI metrics (not due to partial volume effects, but due to degenerative processes).

Conclusion

In conclusion, we demonstrated a significant relationship between atrophy rate and disease progression in our cohort of ALS patients. In addition, our results suggest that spinal cord cross-sectional area and MTR are more reliable markers of longitudinal neurodegeneration in the spinal cord compared to DTI metrics such as FA or radial diffusivity. However the somewhat lower sensitivity of diffusion MRI might be overcome thanks to the current improvements in hardware, acquisition and processing techniques. Further longitudinal studies in a larger population are needed to validate atrophy rate of the spinal cord as a valid surrogate marker of disease progression in ALS.

Study 5 -Cervical spinal cord atrophy profile in adult SMN1-linked SMA

Background and objectives

In this part of my thesis, we analyze the extent of neurodegeneration in another MND, i.e. adult SMN1-linked SMA that shares a common feature of LMN loss with ALS. We investigated the topography of LMN degeneration along the spinal cord in SMA. The clinical data with a classical proximal predominance of the deficit suggests that LMN degeneration may predominate in spinal levels innervating proximal muscles. Whether it corresponds to a selective vulnerability of motor neuron pools depending on their location within the spinal cord and/or to peripheral factors, particularly related to different muscle characteristics, remains unknown. Nevertheless, there is no post-mortem study in SMA patients that studies the distribution of motor neuron loss along the spinal cord and establishes correlations with clinical deficits.

New developments in spinal cord MRI provide a unique opportunity to produce an in-vivo index reflecting LMN loss, as it has been recently shown in our studies of ALS patients (Cohen-Adad et al., 2013; Study 4). In our previous work, we developed and validated a segmentation method that accurately quantifies spinal cord cross-sectional area and volume at large cervical and thoracic spinal cord levels (Study 1 and 2). Here we tested the hypothesis that the classical proximal predominance of the motor deficit may be due to the predominant degeneration of motoneurons at specific spinal cord levels. We therefore quantified, using spinal MRI, the degree of atrophy at different cervical levels as well as the motor deficit seen in these patients. For this purpose, a surface-based morphometry method was used for the first time to produce a three-dimensional cord atrophy profile along the cervical spinal cord and to establish comparisons with the distribution of weakness in SMA patients.

Material and Methods

Eighteen SMN1-linked SMA patients (types: 5 IIIa, 10 IIIb and 3 IV) and 18 age/gender-matched healthy volunteers were recruited. Patients were scored on manual muscle testing, motor function measure (Bérard et al., 2005), and the ALSFRS-R. The spinal cord was imaged using high-resolution 3D T2-weighted sequence from 3T MRI system. Surface-based morphometry technique was used to produce a three-dimensional cervical spinal cord atrophy profile (Morra et al., 2008). Radial distance (Lundell et al., 2011;Morra et al., 2008), and cord cross-sectional area measurements in SMA patients were compared to those in controls and correlated with strength and disability scores.

Materials and Methods

Eighteen SMN1-linked SMA patients type III/V and 18 age/gender-matched healthy volunteers were included. Patients were scored on manual muscle testing and functional scales. Spinal cord was imaged using 3T MRI system. Radial distance (RD) and cord cross-sectional area (CSA) measurements in SMA patients were compared to those in controls and correlated with strength and disability scores.

Results

CSA measurements revealed a significant cord atrophy gradient mainly located between C3 and C6 vertebral levels with a SCA rate ranging from 5.4% to 23% in SMA patients compared to controls. RD was significantly lower in SMA patients compared to controls in the anterior-posterior direction with a maximum along C4 and C5 vertebral levels (p-values < 10 -5 ). There were no correlations between atrophy measurements, strength and disability scores.

Conclusions

Spinal cord atrophy in adult SMN1-linked SMA predominates in the segments innervating the proximal muscles. Additional factors such as neuromuscular junction or intrinsic skeletal

Introduction

Spinal muscular atrophy (SMA) is a group of degenerative diseases affecting lower motor neurons [1]. SMA diseases are classically separated in proximal and distal forms [1]. The proximal form of SMA is mainly due to deletions or mutations in the telomeric copy of the survival motor neuron gene-1 (SMN1) [2] SMA III is a milder form of SMA starting after the achievement of walking and is subdivided in SMA IIIa with an onset before 3 years and SMA IIIb after 3 years. Eventually, SMA IV refers to an onset in adults (>18 years) [3]. The reason of the classical proximal predominance of the deficit is unknown and its elucidation would provide an important clue to understand the physiopathology of the disease [4]. New developments in spinal cord MRI provide a unique opportunity to investigate in vivo cord atrophy and to produce an index reflecting lower motor neurons loss, which has never been reported in any SMA type. In ALS, another motor neuron disease that combines lower and upper motor neuron degeneration, cord atrophy measurement reflected grey matter loss [5,6]. We tested here the hypothesis that the classical proximal predominance of the motor deficit in SMA may be due to predominant degeneration of lower motor neurons at specific spinal cord levels. We therefore quantified, using spinal MRI and for the first time a surface based morphometry technic, the degree of atrophy at different cervical levels and establish comparisons with the distribution of weakness in SMA patients.

Material and Methods

Subjects

Clinical examination

Muscle strength was measured by manual muscle testing (MMT) and performed in all patients by the same experienced neurologist (P.-F. P. among the authors) using the Medical Research Council (MRC) scale (0-5) [7]. Particular care was taken to have the patients relaxed before examinations since it was noted that patients with SMA got fatigue easily, which biased muscle strength measurements. MMT subscores were calculated by grouping muscles according to their motor column locations at cervical spinal cord segments: C5 (shoulder abduction), C6 (elbow flexion), C7 (wrist flexion/extension), C8 (abduction of the 1 st finger and distal flexion of the 3 rd finger). Motor Function Measure (MFM) scale was performed [8]. MFM provides a measurement of the motor capacity by three functional indexes: standing and transfer (D1), axial and proximal motor function (D2) and distal motor function (D3). In addition, SMA patients were scored on the Revised ALS Functional Rating Scale (ALSFRS-R) [9]. An arm ALSFRS-R subscore (handwriting, cutting food and handling utensils), and a leg ALSFRS-R subscore (walking, climbing stairs) were calculated (S1 Dataset).

MRI acquisition

Subjects were positioned head-first supine, with a 2-centimeter-thick pillow to lift the head and no pillow below the neck. This strategy was used to limit the natural cervical cord lordosis at around C3-C4, i.e., excessive cord curvature in the antero-posterior (A-P) direction. Subjects were positioned as comfortable as possible, centered in the scanner's reference and were systematically asked not to move during the acquisition in order to minimize motion artifacts.

Scans were performed using a 3T MRI system (TIM Trio 32-channel, Siemens Healthcare, Erlangen, Germany). The spinal cord was imaged using a three-dimensional (3D) T2-weighted turbo spin echo acquisition with a slab-selective excitation pulse (SPACE sequence: sampling perfection with application optimized contrasts using different flip angle evolution). Imaging parameters were: voxel size = 0.9×0.9×0.9 mm 3 ; Field of View (FOV) = 280×280 mm 2 ; 52 sagittal slices; Repetition time (TR)/ Echo time (TE) = 1500/120ms; flip angle = 140°; generalized autocalibrating partially parallel acquisition (GRAPPA) with acceleration factor R = 3; turbo factor = 69; acquisition time was 6 min [5,6].

Data processing

Preprocessing. Data were corrected for non-uniformity intensity using Minc-Toolkit N3 [10]. Two landmarks have been manually defined in the sagittal slice where the spinal cord was the most median in the FOV by an experienced operator on spinal cord MRI segmentation techniques (M.-M. E. M., 5 years of experience, among the authors), to delimit the cervical spinal cord (i.e. from the upper limit of the odontoid process of the C2 vertebrae to the middle vertebral body C7/T1) [11,12]. Data were cropped and resampled to a voxel size of 0.3×0.3×0.3 mm 3 using 3D cubic interpolation in order to maximize segmentation accuracy [12][13][14]. Fig 1A shows a preprocessed mid-sagittal section in an SMA patient.

Segmentation. Data were segmented using a double threshold-based method (DTbM), an improved version of the well-established threshold-based method (TbM) [12,14,15]. The same experienced operator as previously, adjusted visually rare poor segmentations using TbM and, when needed, manually [12]. Fig 1B shows an example of segmentation result in an SMA patient.

Cord standardization. Spinal cord masks resulting from segmentation were straightened, then standardized in length by rescaling them to the median cord length (median length across subjects = 413 slices) using 3D nearest neighbor interpolation, leading to a straight and well centered spinal cord masks in the A-P and right-left (R-L) directions in the FOV [11,12]. Fig 1C and1D show an example of cord and mask straightening results in an SMA patient.

Radial distance and cross-sectional area measurements. Radiuses from the center of mass of each axial mask to its borders were measured with an angular resolution of 5°by mapping Cartesian coordinates to polar coordinates (one radius every 5°), defined as radial distance (RD) (Fig 1E) [13]. This distance could be seen as a measurement of spinal cord thickness. RD enabled to detect a preferential A-P atrophy direction in spinal cord injuries (SCI) at C2 vertebral level that correlated with the clinical status of SCI patients, represented by the ASIA motor and sensory [13]. RD has also been used to spatially localize the hippocampus diffuse atrophy in Alzheimer patients, a tissue that has a geometry resembling to a bent elliptic cylinder as for the spinal cord [16]. Cross-sectional area (CSA) was also computed. CSA was defined as the cord area in mm 2 covered by each axial mask along the standardized spinal cord images (S1 Dataset).

Statistical analysis

Statistical analysis was performed using Matlab 1 (The Mathworks Inc, MA, USA).

Comparison between distal and proximal muscles strength. Wilcoxon signed rank test was performed to test the difference in strength between proximal (MMT C5 spinal level; shoulder abduction) and distal muscles (MMT C8 spinal level; abduction of the 1 st finger and distal flexion of the 3 rd finger) in SMA patients, a significance level alpha = 0.05 was used.

Comparisons of MR measurements between patients and controls. RD and CSA along the standardized spinal cord images were compared between SMA patients and controls using permutation test (one side, 100.000 permutations) [16]. Permutation tests measure the distribution of atrophy features represented by RD or CSA decrease in SMA patients compared to controls that would occur by chance if patients and controls were randomly assigned to groups [17]. The high number of permutations 100.000 was chosen to control the standard error of the omnibus probability p (SEp) [18]. Given the high number of computed comparisons (72×413 permutation tests for RD comparisons and 413 permutation tests for CSA comparisons), a supra-significance level alpha = 10 -3 was used [13,16]. The previously constructed template [12] was used as a visualization support for the 3D p-values profile resulted from RD comparisons between SMA patients and controls (S1 Dataset). Mean patients CSA was subtracted from mean controls CSA then normalized by mean controls CSA to obtain cord atrophy rate along the cervical spinal cord (in %).

Correlations between MR measurements, clinical disability and disease duration. Spearman's rank correlation coefficient was used to investigate correlations between RD and CSA along the cervical spinal cord and disease duration, MMT, MFM and ALSFRS-R subscores (non-normally distributed and ordinal data). Due to the high number of comparisons (413 correlation tests), a supra-significance level alpha = 10 -3 was used [13,16]. In addition, CSA gradient from C4 to C7 vertebral levels, corresponding mainly to C5-C8 spinal segments [19] was correlated, using Spearman's rank correlation coefficient, with MMT variation from C5 to C8 spinal segments. Rostro-caudal CSA gradient and proximal-distal muscles strength variation were represented by the slop of the straight line that best fits CSA curve and MMT subscores, resulting from a linear regression model.

Results

Demographics and clinical features

Eighteen SMN1-linked SMA patients and 18 age and gender matched healthy volunteers have been recruited. Population demographics and clinical futures in SMA patients are summarized in Table 1. MMT revealed diffuse weakness in the upper limbs involving the muscles innervated by C5 to C8 spinal segments. There was no significant difference between proximal (C5) and distal muscle strength (C8) (Wilcoxon signed rank test; p-value = 0.89) in SMA patients.

Study of spinal cord atrophy

Comparisons between patients and controls. Fig 2 shows the 3D p-values profile resulting from RD comparisons between SMA patients and controls. RD was significantly lower in SMA patients compared to controls in the anterior-posterior direction and mostly located between C3 and C6 vertebral levels, corresponding to C4-C7 spinal segments, with a maximum along C4 and C5 vertebral levels, corresponding to C5-C6 spinal segments, (p-values < 10 -5 ) [19].

Fig 3A and3B show CSA profile from SMA patients and controls and cord atrophy rate along the cervical spinal cord, respectively. CSA showed a significant atrophy gradient mainly located between C3 and C6 vertebral levels with a cord atrophy rate ranging from 5.4% to 23%. CSA difference between SMA patients and controls significantly increased from C2 to C3 vertebral level and remained stable along C4 vertebral level with a mean cord atrophy rate equal to 20%. After this plateau, CSA difference decreased slowly and remained significant to the upper part of C7 vertebral level and was no longer significant beyond.

Correlations between MR measurements, clinical disability and disease duration. There were no correlations between RD, CSA along the cervical spinal cord and clinical disability subscores MMT, MFM and ALSFRS-R, nor between RD, CSA and disease duration (p-values > 10 -3 ) (S1-S5 Figs). No correlation was detected between rostro-caudal CSA gradient and proximal-distal muscles strength variation (p-value = 0.35).

Discussion

The main results of the present study are: 1) a three-and two-dimensional cervical spinal cord atrophy profile can be established in SMA types III and IV; 2) the A-P predominance of atrophy suggests that RD, in addition to CSA, could be considered as an index of motor neurons loss in the anterior horns; 3) there were no correlations between RD/CSA and the degree or the topography of clinical deficits.

Spinal cord atrophy was quantified using an accurate segmentation method, DTbM [12]. Segmentation accuracy was increased by adjusting rare poor segmentations using TbM and, when needed, manually [12,14]. Furthermore, a supra-significance level alpha = 10 -3 was used to minimize false positive results due to error in RD and CSA estimations [13,16]. The combination of high segmentation accuracy [12] cord standardization procedure that allowed groups' comparison for large spinal cord segments [11,12] enabled producing for the first time an accurate 3D cervical spinal cord atrophy profile in SMA patients using RD [12,16]. RD analysis showed preferential cord atrophy in the A-P direction of the spinal cord between C3 and C6 vertebral levels, corresponding to C4-C7 spinal segments [19]. The apparent atrophy at dorsal side of the spinal cord was probably an indirect consequence of anterior horns atrophy. In fact, the large atrophy in the ventral region produces a displacement of centers of mass of axial masks towards the dorsal side of the spinal cord. Thus, the measured radiuses from the center of mass decreased in both dorsal and ventral regions in SMA patients, which may explain the visible atrophy at both sides of the spinal cord. It cannot also been ruled out that the atrophy of the dorsal column may participate to some extent. Besides post-mortem and histological studies that reported lower motor neuron loss in SMA patients at the cervical cord level [20][21][22], two autopsy cases of SMN1-linked SMA patients type III showed a loss of the fasciculus gracilis myelinated fibers [22,23].

The fact that atrophy predominates in the C5 and C6 spinal segments fits with the classical proximal predominance of muscle weakness in SMN1-linked SMA. Our results are also in agreement with a study in delta 7 SMN mice that compared the loss of motor neurons at different spinal cord levels [24]. The authors showed that the reduction of the mean somatic area of motor neurons was maximal at the C5-C6 spinal levels innervating proximal forelimb muscles and was less severe, and not statistically significant rostrally at the C3 level (phrenic motor neurons) and caudally at the C8 level (forelimb distal muscles).

We showed that RD and CSA were not correlated with clinical deficits. First, we did not find correlations between atrophy at a given spinal level and the deficit in the corresponding innervated muscles. Second, the gradient of spinal cord atrophy did not mirror the distribution of weakness in our series of patients. In addition, MMT at the upper limbs showed the presence of distal weakness, which is in accordance with analysis of the literature in SMN1-linked SMA patients type III/IV [25][26][27][28][29]. Interestingly, a study in a rodent SMA model showed that muscle denervation was located throughout the body involving both proximal and distal regions [30].

In our series, the absence of correlation between SCA and weakness could be related to methodological limits, mainly the small number of patients in this rare disease and a lack of sensitivity of MMT for weakness quantification. Despite of these limitations, our findings are in line with the complexity of SMA pathogenesis, which is related to multiple factors, involving motor neuron cell bodies, motor axons and muscles, converging in affecting the integrity and function of the neuromuscular unit leading to muscle deficits [24,30,31]. Particularly, studies in various SMA mouse models pointed out that the disease process may be initiated at the neuromuscular junctions with reduced transmitter release, smaller and immature endplates and neurofilament accumulation at presynaptic terminals [23,[32][33][34]. A recent study using repetitive nerve stimulation showed a decrement in nearly one half of SMA patients types II and III [35]. Such evidence for dysfunction of neuromuscular junction was detected both in distal and proximal muscle groups of the arm and was predominantly postsynaptic. Interestingly, recent findings in different SMA animal models have highlighted the contribution of intrinsic skeletal muscle defects, with disruption of the myogenic program associated with a decrease in myofiber size and an increase in immature myofibers [36]. A study in an SMA feline model provided convincing evidence that motor neuron degeneration begins distal to the cell body and proceeds retrogradely (« dying-back process ») [37].

From a more clinical perspective, the sensitivity of MRI suggests that quantification of spinal cord atrophy could be a potential biomarker of motor neurons loss in SMA, as previously shown in ALS [5,6,38]. There is an unmet need for surrogate markers of disease progression in SMA since clinical scales mildly change over time, especially in SMA types III and IV, in which motor disability worsen very slowly [25,28,39]. However, longitudinal studies are needed to evaluate if RD and CSA are sensitive to change over time, as shown for CSA in ALS [6] and could be used as a tool to monitor the effect of investigational therapies. These spinal cord MRI metrics are also applicable to other lower motor neuron diseases such as monomelic atrophy (Hirayama disease) to localize local spinal cord flattening and atrophy [40,41].

Improvement in MRI techniques are promising to increase the sensitivity of the method particularly at the lumbar spinal cord level which remains technically challenging due to increased physiological motions compared to the cervical level [42]. In the future, the development of high-resolution MRI sequences offers the opportunity to specifically measure grey matter loss [42,43]. However, the absence of correlations between MRI measurements and clinical deficits suggests that the combination with other methods, particularly electrophysiology [35,44], muscle imaging [44] or electrical impedance myography [45], may be more suitable to measure disease progression in respect with the complexity of the disease.

Conclusions

This study is a proof of concept that advanced MRI methods can provide indexes of motor neurons loss in the anterior horns in SMN1-linked SMA. Thanks to theses methods, we were able to demonstate that atrophy predominates in the segments innervating the proximal muscles in SMA. In the future, awareness about the potential of advanced MRI methods in this research field should foster collaborative studies that are needed to include larger number of patients and increase the statistical power in transversal and longitudinal studies. 3.9±1.2 min/subject (mean ± SD), which is two time faster than Losseff's method. Our method was rigorously evaluated both qualitatively by visual evaluation made by an experienced neuroradiologist, and quantitatively at C2 vertebral level, the cervical and thoracic regions in 3T T2-weighted data from healthy subjects and patients with ALS, SMA and SCI. DTbM outperformed active surface method (ASM) [START_REF] Horsfield | Rapid semi-automatic segmentation of the spinal cord from magnetic resonance images: application in multiple sclerosis[END_REF], the second most used segmentation method to quantify atrophy in MS after Losseff's method (De leener et al., 2016a), for both quantitative and qualitative accuracy evaluation. Our method showed similar accuracy to Losseff's method and higher accuracy than ASM.

Supporting Information

Despite its high accuracy, our method failed to outline the spinal cord in regions of large areas of T 2hyperintensity, large regions of spinal canal narrowing with limited CSF space (one SMA patient), as well as at the lesion level in four SCI patients with T 2 -hypersignal and atrophy of the spinal cord and narrow spinal canal. Another limitation of our study is that we did not have the opportunity to conduct a scan-rescan evaluation of DTbM. However, DTbM is based on the well-established Losseff's method, which showed low scan-rescan variability (Leary et al., 1999). Thus, DTbM is expected to have the same order of variability as Losseff's method. In addition, we designed a data normalization pipeline that includes DTbM, enabling the construction of an accurate straight 3T T2-weighted cervical spinal cord template using MRI data from 59 healthy volunteers. Parallel to our methodological development, De leener et al., (2014) proposed an automated segmentation method that showed lower accuracy and DTbM. The same group proposed a standardization method that leads to an error in cross-sectional area estimation of 16% (4.8% and 6.5% for DTbM at the cervical and thoracic levels, respectively), which would make the method too inaccurate for atrophy quantification in the pathological context Fonov et al. (2015).

Grey and white matter could not be differentiated in the acquired images due to insufficient contrast inside the spinal cord. However, atrophy may predominate in a particular direction in neurodegenerative diseases and trauma. Indeed, ALS is characterized by cell loss in the anterior horn and the CST degeneration (Wijesekera and Leigh, 2009). The CST degeneration and loss of alpha motor neurons may result in atrophy in the lateral and ventral direction, respectively. SMA is characterized by degeneration of cell bodies in the anterior horn (Katsuno et al., 2010), which potentially induces a pronounced ventral atrophy. In SCI patients, more pronounced atrophy was reported in the anteroposterior direction than in the left/right direction at C2 vertebral level (Lundell et al., 2011). This preferential direction of cord atrophy may be detected in group analysis using the surface-based morphometry method (Morra et al., 2009;Lundell et al., 2011). The hypothesis of a preferential ventral direction in SMA patients was confirmed using our data processing tools combined, for the first time at the spinal cord level, with the surface-based morphometry method (Study 5). Parallel to our atrophy investigation, Taso et al. (2015) proposed mapping spinal cord white and grey matter alterations occurring with age using TBM method. The authors showed that regional spinal cord atrophy was mostly localized in anterior horns in elderly healthy subjects. This method holds great promise for investigating motor neuron loss occurring in MND. However, due to technical and time constraints of our imaging protocol, we could not acquire such high resolution MR images enabling to investigate more specifically regional white and grey matter atrophy in MND. The same method could provide supporting evidence of motoneurons loss at the upper cervical spinal cord in progressive MS pathology [START_REF] Schlaeger | Spinal cord gray matter atrophy correlates with multiple sclerosis disability[END_REF][START_REF] Schlaeger | Association Between Thoracic Spinal Cord Gray Matter Atrophy and Disability in Multiple Sclerosis[END_REF][START_REF] Vogt | Lower motor neuron loss in multiple sclerosis and experimental autoimmune encephalomyelitis[END_REF][START_REF] Davison | Multiple sclerosis and amyotrophies[END_REF]. In SCI and spinocerebellar ataxia type 3 patients, TBM could be used to investigate the regional contribution of white and grey matter atrophy in the preferential anterior-posterior cord flattening at C2 vertebra level (Lundell et al., 2011;[START_REF] Fahl | Spinal cord damage in Machado-Joseph disease[END_REF].

Multi-parametric MRI studies in MND

Lesions of the spinal cord due to neurodegenerative diseases lead to major motor and sensory consequences whose severity depends on their extent. In our studies in MND, we attempt to establish the extent of the spinal lesions as precisely as possible by using multi-parametric MRI as a complementary approach to clinical and electrophysiological examinations.

In a previous study in ALS patients (Cohen-Adad et al. 2013), we showed in the spinal cord, by using DTI and MT imaging, that the dorsal column, i.e. those pathways conveying peripheral sensory information to supraspinal structures were altered. Dorsal column was also involved in a selected subgroup of 12 patients with an early onset of disease (median/maximum disease duration 9/12.6 months), which suggested a sensory involvement at an early stage of the disease. In that respect, we coupled spinal DTI and electrophysiological recordings to further investigate the anatomofunctional properties of sensory pathways in ALS patients and to give more evidence of their involvement. The selected patients had no sensory symptoms or signs and medical conditions associated with peripheral neuropathy. This combined approach revealed sub-clinical sensory defects in 85% of ALS patients, while separated methods revealed abnormal values in 60%. Our results strongly suggest peripheral and central sensory defects, which have been underestimated in previous studies. Together with our previous MRI study, these results demonstrated that advanced spinal cord MRI techniques, combined with clinical and electrophysiological measurements, enabled us to investigate motor and extra-motor system involvement in ALS. Our results suggested that sensory afferent pathways to spinal and cortical structures might be already affected at an early stages in ALS, and thus that sensory alterations may indeed participate to ALS pathogenesis. In this respect, a recent electrophysiological study from our group in the same ALS patients suggested that sensory afferents and interneurons involvement leads to motoneurons hyper-excitability, which may contribute to their degeneration (Sangari et al., 2016). The latter result is in line with several studies in humans and animal models, suggesting that motoneurons loss is probably not due to their selective vulnerability but a consequence of converging factors causing their degeneration [START_REF] King | Excitotoxicity in ALS: Overstimulation, or overreaction?[END_REF]. These factors did not affect exclusively motoneurons but comprise several neurons and cells that are interconnected with; i.e., sensory neurons, interneurons, Beyond the topographical extension of the lesions, we investigated their progression over time.

Our longitudinal study suggested that LMN degeneration, as reflected by atrophy (Cohen-Adad et al., 2013), was the major determinant of the disease progression. In addition, our study suggests that white matter lesions in the CST and also in the dorsal column (sensory afferents; unpublished data), may be relatively constant during the symptomatic period and, by contrast, clinical progression is mainly dependent on the extent of anterior horn cell degeneration, as assessed by correlations between atrophy changes and functional deterioration. In this respect, a recent study at the brain level suggested that axonal lesions, reflected by DTI changes along the CST, remain relatively constant in the symptomatic period contrasting with the extension of grey matter lesions, as assessed by VBM, and further supported by the lack of change over time in UMN clinical score in nearly all ALS patients (Menke et al., 2014).

Altogether, these results suggest that molecular mechanism underlying degeneration of axons and neuronal cell bodies may not progress at the same rate during the course of the disease. We indicate bellow a putative model of the dynamic of axonal and grey matter degeneration (Figure 16). Using MRI, we analyzed the topographical extension of lesions in adult SMN1-linked SMA that share a common feature of LMN loss with ALS. The key findings of lesions extent analysis in SMA patients may be summarized as follows: first, we showed that cord atrophy predominates in the spinal segments innervating the proximal muscles, in agreement with a study in delta 7 SMN mice that compared the loss of motor neurons at different spinal cord levels [START_REF] Errico | Selective vulnerability of spinal and cortical motor neuron subpopulations in delta7 SMA mice[END_REF]. Second, we did not find correlations between atrophy at a given spinal level and the deficit in the corresponding innervated muscles. In addition, the gradient of spinal cord atrophy did not mirror the distribution of weakness in our series of patients. The fact that atrophy predominates the spinal segments innervating the proximal muscles may have fitted with the general assumption that SMA is characterized by proximal muscle weakness. However, muscle strength in the upper limbs showed a more diffuse deficit with the presence of distal weakness, which is in accordance with analysis of the literature in SMN1-linked SMA patients type III/IV (Carter et al., 1995;Gardner-Medwin et al., 1967;Meadows et al., 1969; Hausmanowa-Petrusewicz and Fidzianska-Dolot, 1984; Piepers et al., 2008). Finally, our findings are in line with the concept of SMA as a multi-system disorder, which is related to multiple factors, involving motor neuron cell bodies, motor axons and muscles, converging in affecting the integrity and function of the neuromuscular unit leading to muscle deficits (Hamilton and Gillingwater, 2013

Perspectives

From a more clinical perspective, the sensitivity of MRI suggests that quantification of spinal cord atrophy could be a potential biomarker of motor neuron loss in both SMA and ALS. There is an unmet need for surrogate markers of disease progression in SMA and ALS. In SMA, clinical scales mildly change over time, especially in SMA types III and IV, in which motor disability worsen very slowly (Carter et al., 1995;Piepers et al., 2008;Werlauff et al., 2012). In both conditions, longitudinal studies are mandatory to evaluate if multi-parametric MRI metrics are sensitive enough to change over time and thus could be used as tools to monitor the effect of investigational therapies. Nevertheless, improvements in MRI techniques are promising to increase the sensitivity of these metrics, particularly at the lumbar spinal cord level, which remains technically challenging due to increased physiological motions compared to the cervical level. In the future, the development of high-resolution MRI sequences will offer the opportunity to specifically measure grey matter loss (Taso et al., 2014(Taso et al., , 2015;;[START_REF] Schlaeger | Association Between Thoracic Spinal Cord Gray Matter Atrophy and Disability in Multiple Sclerosis[END_REF]Yiannakes et al., 2014). The combination of multi-parametric MRI with other methods, particularly electrophysiology and advanced clinical examinations, may be more suitable to measure disease progression with respect to the complexity of MND. A new project (SPINE 2; Primary investigator: Pierre-François Pradat; Sponsor: IRME (Institut de Recherche sur la Moelle épinière et l'Encéphale)) that includes advanced MRI techniques, benefiting from recent advances in the field of MRI, combined with electrophysiology and tools for quantification of muscle weakness is ongoing. The aim of SPINE 2 project is to pursue our investigation of lesion spread in ALS, SMA and another MND, X-linked spinal bulbar muscular atrophy, at both cervical spinal cord and brain level.

Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a progressive alteration in the upper, or cortical, motor neurons and lower motor neurons, located in the spinal cord and brainstem. Due to its clinical heterogeneity and the lack of biological markers to diagnose ALS, the delay between the first symptoms and the diagnosis is evaluated at 9-13 months [1]. There is an unmet need to find specific and early biomarkers to help to diagnose and characterize phenotype or progression [2].

Signs of central motor neuron degeneration are often difficult to detect in clinical practice, justifying the interest for objective neuroimaging markers of upper motor neuron (UMN) involvement. It has been estimated that clinical UMN signs are absent at first examination in 7 to 10% of patients who further develop full-blown ALS [3,4]. The diagnostic delay is increased in patients who present isolated lower motor neuron (LMN) signs [3]. It is needed to rule out other LMN pathologies such as spinal muscular atrophy (SMA), bulbospinal muscular atrophy (Kennedy disease), or multifocal neuropathies with conduction blocks [1]. The apparition of central signs during the follow-up may allow confirming the diagnosis of ALS but this is not the case in all patients, notably because the severity of peripheral signs can mask central signs.

Neuroimaging methods allow investigating in vivo the extent of neurological systems degeneration. There is a growing body of evidence demonstrating that ALS is a multisystem neurological disorder. Autopsy studies have shown that degeneration of central nervous system structures is not restricted to the primary motor cortex and the pyramidal tract [5][6][7][8]. In addition to motor signs, cognitive signs are detected by neuropsychological tests in about 50% of patients with sporadic ALS and typical frontotemporal dementia (FTD) occurs in approximately 10% of the patients [9,10]. Association of ALS and FTD occurs in the majority of C9ORF72-linked familial ALS (FALS) [11,12]. Atypical clinical features can be associated, defining "ALS plus" [1] syndromes with signs and symptoms involving the sensory (particularly in SOD1-linked FALS) [13][14][15][16][17][18][19][20][21][22], extrapyramidal [14,[23][24][25][26][27][28][29][30][31], cerebellar [23,32], ocular [33,34], and autonomic [35] systems.

In this paper, we will review the main findings obtained with different modalities of neuroimaging in ALS. We will especially focus on magnetic resonance imaging (MRI) studies and particularly on spinal cord neuroimaging that has shown great developments in the last few years.

Magnetic Resonance Imaging

2.1. Conventional Magnetic Resonance Imaging. Beyond its role to exclude several "ALS-mimick" syndromes [1], abnormalities suggestive of upper motor neuron involvement are sometimes detected. Several studies, using various modalities (T2 * , fluid-attenuated inversion recovery (FLAIR) or fast spin echo proton density-weighted imaging), have shown hyperintensity in the white matter along the corticospinal tract, from the centrum semiovale to the brainstem [36][37][38][39][40][41][42][43][44][45][46]. However, such abnormalities are rare, nonspecific, not readily quantifiable, and do not correlate with disease severity or rate of progression [47]. A cortical atrophy, which is predominant in the frontal region, and a characteristic T2 * or FLAIR hypointensity at the level of the primary motor cortex have been described in the literature but are exceptionally detectable in clinical practice [39,48].

2.2. Advanced MRI. Diffusion-based neuroimaging methods allow evaluating the degeneration of white matter fiber bundles. Diffusion results from the random movement of molecules in vivo. Diffusion of water in structures such as the cerebrospinal fluid (CSF) or grey matter is isotropic (identical in all directions), whereas in white matter it occurs preferentially along the axis of orientation of the fiber bundles. The application of diffusion gradients in several directions results in diffusion tensor imaging (DTI). An anisotropy map can thus be obtained, which provides information about the microstructural organization of the white matter.

A study published in 1999 demonstrated a decrease in fractional anisotropy (FA) in the intracranial portion of the corticospinal tract (subcortical white matter, internal capsule, and brainstem) [49]. It has been confirmed by numerous other studies [50][51][52][53][54][55][56][57][58][59][60]. Abnormalities have been observed in patients who had no UMN signs at the time of MRI investigation but developed pyramidal tract symptoms later in the course of their disease, suggesting that DTI could contribute to earlier diagnosis of ALS in patients with pure LMN involvement [51]. It has been suggested that a decreased fractional anisotropy (FA) in the corpus callosum (CC) was a good DTI marker in patients with ALS [61]. These changes may correspond to degeneration of transcallosal fibers passing between primary motor cortices. Another study showed that FA in motor-related regions of the CC is more affected than in other CC areas in ALS patients [62].

Tractography is a method based on DTI that enables reconstruction of the three-dimensional geometry of the pyramidal tract [63] and allows establishing an FA profile along the pyramidal tract [64]. A tractography study showed that the decreases in FA are largely limited to the precentral areas in patients with ALS [52]. Using tractography to segment the corticobulbar tract, lower FA was measured in patients with bulbar-onset versus limb-onset disease [65].

By using a voxel-by-voxel approach, which consists of comparing groups of patients without an a priori predefined region of interest, our team and other groups have showed that abnormalities on DTI were detectable outside the primary motor regions, thereby confirming that ALS is a multisystem degenerative disorder [53]. Diffuse lesions have also been observed using voxel-based morphometry (VBM), a technic allowing automated segmentation and quantification of grey and white matter volumes to study regional differences [66]. Recently authors used high-resolution T1weighted imaging data for model-based subcortical registration and segmentation to explore the involvement of subcortical structures [67]. They used vertex-wise statistics that provide quantitative, visual, surface-projected information about the shape of the various subcortical structures. This analysis revealed changes affecting the basal ganglia including the superior and inferior aspects of the bilateral thalami, the lateral and inferior portion of the left hippocampus, and the medial and superior aspect of the left caudate (Figure 1). The authors conclude that dysfunction of frontostriatal networks is likely to contribute to the unique neuropsychological profile of ALS, dominated by executive dysfunction, apathy, and deficits in social cognition.

The discovery that the presence of a hexanucleotide expansion in C9orf72 gene [11,12] was associated with FTD in ALS led to specific neuroimaging investigations. A recent study used tract-based spatial statistics of multiple white matter diffusion parameters, cortical thickness measurements, and VBM analyses in C9orf72-negative patients with ALS carrying the C9orf72 hexanucleotide repeat expansion [68]. The result is that extensive cortical and subcortical frontotemporal involvement was identified in association with the C9orf72 genotype, compared to the relatively limited extramotor pathology in patients with C9orf72-negative ALS [67]. In contrast, a DTI study showed that patients with SOD1 gene-linked FALS showed less extensive pathologic white matter in motor and extramotor pathways compared with patients with sporadic ALS [69].

In clinical practice, the diagnostic value of FA measurement remains limited mainly because of the overlap between the values measured in ALS patients and those in control subjects. In one study, measurement of FA in the internal capsule to detect central motor neuron lesions compared with healthy subjects had a sensitivity of 95%, but the specificity was only 71%, with a positive predictive value of 82% [46]. A recent individual patient data (IPD) meta-analysis using corticospinal tract data suggested that the diagnostic accuracy of DTI lacks sufficient discrimination [59]. Of 30 identified studies, 11 corresponding authors provided IPD and 221 ALS patients and 187 healthy control subjects were available for the study. The pooled sensitivity was 0.68 (95% CI: 0.62-0.75), and the pooled specificity was 0.73 (95% CI: 0.66-0.80).

Spinal Cord Imaging.

Spinal MRI has the advantage to investigate the two motor system components that are involved in ALS, that is, the lower and upper motor neuron. Although widely applied to the brain, advanced methods such as DTI are challenging at the spinal level because of (i) the small size of the cord relative to the brain (∼1 cm diameter in the human) requiring higher spatial resolution and thus decreasing the signal-to-noise ratio, (ii) physiological motions (respiration, cardiac) that may bias anisotropic diffusion coefficient estimation and create ghosting artifacts [70][71][72], (iii) partial volume effects that are more problematic in the cord due to the surrounding cerebrospinal fluid [73], (iv) chemical-shift artifacts arising from the epidural fat and other nearby structures, and (v) geometric distortions arising from magnetic field inhomogeneities in nearby intervertebral disks and lungs. The latter point is particularly challenging in diffusion MRI since usual sequences based on echo planar imaging (EPI) are very sensitive to such artifacts [74,75]. In past years, with the development of new methods, such as cardiac and respiratory gating [75][76][77][78], it has been shown that DTI and magnetization transfer (MT) imaging were feasible to detect changes in the spinal cord in ALS [79][80][81]. In a recent study, we have shown that abnormalities in the spinal cord using a multiparametric MRI approach combing DTI, MT ratio (MTR), and atrophy measurements correlated with functional impairment [79]. In this study, local spinal cord atrophy was correlated with muscle deficits and with the motor evoked potential amplitude measured by transcranial magnetic stimulation (TMS), an index of LMN dysfunction. It suggests that regional atrophy is a sensitive biomarker of motor neuron loss in the anterior horns of the spinal cord.

Conversely, DTI and MTR changes in the corticospinal tract correlated with the higher facilitation motor threshold measured by TMS, a parameter that reflects the functionality of the pyramidal tract. Interestingly, changes of DTI metrics demonstrated a subclinical involvement of sensory pathways.

Recently, new acquisition strategies have been proposed to overcome the inherent difficulties in diffusion-weighted imaging of the spinal cord. A promising high-resolution DWI sequence (Syngo RESOLVE) has been proposed [82].

The RESOLVE sequence allows minimization of susceptibility distortions and T2 * blurring. Furthermore, it can be combined with other acquisition strategies such as reduction field-of-view (rFOV) [83][84][85] and parallel imaging [86] to provide fiber tractography in large portions of the spinal cord. Such advances open doors to an accurate quantification of DTI metrics profile along the corticospinal tract or sensory tracts. This is fundamentally needed to clarify some physiopathological aspects of ALS disease, for instance, the dying-back versus dying-forward hypotheses and the possible sensory afferents involvement [79]. In parallel, the new generation of 3 T MRI scanners equipped with 300 mT/m gradients [87,88] provides new exploratory dimensions for white matter microstructures in the spinal cord [89,90]. As showed in vivo as well as ex vivo for the brain, the new MRI scanners improved tissues sensitivity, signal-to-noise ratio (SNR), and spatial and angular diffusion resolution in practical time.

MRI pathological spinal cord studies were mainly focused on white matter integrity where grey matter was not for a great interest until the past few years. This was mainly related to the difficulties in imaging the spinal cord due to low SNR as well as contrastdifference between CSF and white and grey matter at 1.5 T. Passing to higher magnetic field strength (3 T and 7 T) [91][92][93], the construction of adapted coils for spinal cord imaging [94][95][96] and the adaptation of existing sequences [91,97] bring new horizons for spinal cord anatomical explorations. Three recent studies showed the feasibility of white/grey matter imaging and presented reliable tools for anatomical structures characterization in controls [98,99] and multiple sclerosis patients [100] using 3 T MRI-systems. In parallel, one study has showed preliminary results at 3 T of the construction of a probabilistic atlas and anatomical template of the human cervical and thoracic spinal cord that included CSF and white and gray matter [97]. Such atlas can be used for atrophy localization in ALS patients using VBM by adapting the methodology proposed in [START_REF] Valsasina | Spatial normalization and regional assessment of cord atrophy: voxel-based analysis of cervical cord 3D T1weighted images[END_REF] to the case of 3 T MR images (Figure 2).

Such advances bring new perspectives for neurodegenerative diseases and particularly for ALS. Quantifications of white and grey matter degeneration are currently feasible. MRI sequences, image processing, and statistical tools have been already developed [91,97,[START_REF] Valsasina | Spatial normalization and regional assessment of cord atrophy: voxel-based analysis of cervical cord 3D T1weighted images[END_REF][START_REF] El Mendili | Validation of a semiautomated spinal cord segmentation method[END_REF][START_REF] Avants | A reproducible evaluation of ANTs similarity metric performance in brain image registration[END_REF] and are almost ready for use to figure out some of the mechanisms involved in spinal cord tissues degeneration in ALS.

2.4. Functional MRI. Functional MRI (fMRI), by measuring cortical blood oxygen level dependent (BOLD) signal changes, provides a tool to study cortical function and reorganization. Regional modifications in cerebral blood flow have been studied during hand motor tasks [START_REF] Konrad | Pattern of cortical reorganization in amyotrophic lateral sclerosis: a functional magnetic resonance imaging study[END_REF]. Several studies have demonstrated that cerebral activation involved more extensive cortical regions than in control subjects. However, whether it reflects cortical reorganization [START_REF] Konrad | Pattern of cortical reorganization in amyotrophic lateral sclerosis: a functional magnetic resonance imaging study[END_REF]or it is the result of cortical functional adaptation due to peripheral weakness [START_REF] Tempelmann | Functional motor compensation in amyotrophic lateral sclerosis[END_REF] remains a matter of debate. Using a simple hand motor task when the motor deficit is still moderate, a study showed that cerebral activation is correlated with the rate of disease progression suggesting that brain functional rearrangement in ALS may have prognostic implications [START_REF] Poujois | Brain plasticity in the motor network is correlated with disease progression in amyotrophic lateral sclerosis[END_REF]. Unlike the traditional fMRI, the recently developed resting-state functional (rfMRI) techniques avoid potential performance since rfMRI does not require the subjects to perform any task [START_REF] Greicius | Default-mode network activity distinguishes Alzheimer's disease from healthy aging: evidence from functional MRI[END_REF][START_REF] Agosta | Sensorimotor functional connectivity changes in amyotrophic lateral sclerosis[END_REF]. Studies that have been carried out in ALS provide abundant evidence for a reorganisation of various cerebral networks [START_REF] Agosta | Sensorimotor functional connectivity changes in amyotrophic lateral sclerosis[END_REF][START_REF] Douaud | Integration of structural and functional magnetic resonance imaging in amyotrophic lateral sclerosis[END_REF][START_REF] Verstraete | Motor network degeneration in amyotrophic lateral sclerosis: a structural and functional connectivity study[END_REF][START_REF] Jelsone-Swain | Reduced interhemispheric functional connectivity in the motor cortex during rest in limb-onset amyotrophic lateral sclerosis[END_REF][START_REF] Agosta | Divergent brain network connectivity in amyotrophic lateral sclerosis[END_REF][START_REF] Kollewe | Magnetic resonance imaging in amyotrophic lateral sclerosis[END_REF]. Interestingly, one study showed that rfMRI changes correlated with the rate of disease progression and duration [START_REF] Luo | Patterns of spontaneous brain activity in amyotrophic lateral sclerosis: a resting-state fMRI study[END_REF].

Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance spectroscopy (NMRS) allows measuring the neurochemical profile of a particular region of the brain in vivo. The main peak is N-acetylaspartate (NAA), which is considered as a marker of neuronal integrity. Several studies have demonstrated a decrease in NAA [START_REF] Pohl | Proton magnetic resonance spectroscopy of the motor cortex in 70 patients with amyotrophic lateral sclerosis[END_REF][START_REF] Sarchielli | Magnetic resonance imaging and 1H-magnetic resonance spectroscopy in amyotrophic lateral sclerosis[END_REF] and/or ratios of NAA with choline-containing compounds (Cho) and creatine (Cr) [NAA/Cho and NAA/Cr ratios] at the level of the motor cortex of patients with ALS [START_REF] Pohl | Proton magnetic resonance spectroscopy of the motor cortex in 70 patients with amyotrophic lateral sclerosis[END_REF][START_REF] Suhy | Early detection and longitudinal changes in amyotrophic lateral sclerosis by 1H MRSI[END_REF].

For diagnosis applications, studies suggested that NMRS spectroscopy may discriminate between patients with classical ALS and patients with progressive muscular atrophy, a disease confined to LMNs [START_REF] Gredal | Quantification of brain metabolites in amyotrophic lateral sclerosis by localized proton magnetic resonance spectroscopy[END_REF][START_REF] Pioro | Detection of cortical neuron loss in motor neuron disease by proton magnetic resonance spectroscopic imaging in vivo[END_REF]. The diagnostic value is limited because of an overlap between those values in ALS patients and healthy controls. It has been suggested that the combination of N-acetylaspartate (NAA) and myo-inositol may improve the specificity of the test but further studies are needed [START_REF] Kalra | Detection of cerebral degeneration in amyotrophic lateral sclerosis using high-field magnetic resonance spectroscopy[END_REF]. Using a whole-brain resonance spectroscopic imaging approach, NAA showed a significant relationship with disability [START_REF] Stagg | Whole-brain magnetic resonance spectroscopic imaging measures are related to disability in ALS[END_REF]. A cross-sectional study assessing proton NMRS of the cervical spine showed that NAA/Cr and NAA/Myo ratios were reduced in patients with ALS but also in SOD1-positive people at risk for FALS, suggesting that neurometabolic changes occur early in the course of the disease process [START_REF] Carew | Presymptomatic spinal cord neurometabolic findings in SOD1-positive people at risk for familial ALS[END_REF]. Recent advances in high resolution NMRS at 3 T allow direct quantification of GABA in the cortex [START_REF] Zhu | High resolution spectroscopic imaging of GABA at 3 Tesla[END_REF]. Recently, a study in a small number of subjects showed that decreased levels of GABA were present in the motor cortex of ALS patients compared to healthy controls [START_REF] Foerster | Decreased motor cortexaminobutyric acid in amyotrophic lateral sclerosis[END_REF]. It suggests that a loss of inhibition by interneurons may play a role in neurodegeneration through excitotoxicity mechanisms.

Positron Emission Tomography

Positron emission tomography (PET) and monophotonic emission tomography (single photon emission computerized tomography (SPECT)) are nuclear imaging techniques which use various tracers to either reveal neuron dysfunction or investigate a pathogenic mechanism involved in the disease. Using SPECT, several studies have demonstrated a decrease in cerebral blood flow after injection of hexamethyl-propyleneamine oxime labelled with technetium-99 m (99 mTc). They showed abnormalities in the primary motor cortex [START_REF] Waragai | Serial MRI and SPECT in amyotrophic lateral sclerosis: a case report[END_REF][START_REF] Waldemar | Focal reductions of cerebral blood flow in amyotrophic lateral sclerosis: a [99mTc]-d,l-HMPAO SPECT study[END_REF][START_REF] Abe | Reduced isotope uptake restricted to the motor area in patients with amyotrophic lateral sclerosis[END_REF][START_REF] Kalra | Neuroimaging in amyotrophic lateral sclerosis[END_REF], which can also extend in an anterior fashion into the frontal lobes, particularly in patients with associated cognitive problems [START_REF] Habert | Brain perfusion imaging in amyotrophic lateral sclerosis: extent of cortical changes according to the severity and topography of motor impairment[END_REF]. Studies using PET with 2-fluoro-2-deoxy-glucose also showed a variable decrease in cerebral glucose metabolism at rest [START_REF] Hoffman | Cerebral glucose utilization in motor neuron disease[END_REF][START_REF] Hatazawa | Cortical motor-sensory hypometabolism in amyotrophic lateral sclerosis: a PET study[END_REF][START_REF] Kew | Cortical function in amyotrophic lateral sclerosis. A positron emission tomography study[END_REF]. Interestingly, a PET study found not only hypometabolic but also hypermetabolic areas in the brain of sporadic ALS patients, possibly due to increased FDG uptake by astrocytes and/or microglia [START_REF] Cistaro | Brain hypermetabolism in amyotrophic lateral sclerosis: a FDG PET study in ALS of spinal and bulbar onset[END_REF]. In a recent study, the authors report an FDG metabolism study in patients with the C9orf72 mutation compared to nonmutated ALS patients (either with or without dementia) [START_REF] Cistaro | The metabolic signature of C9ORF72-related ALS: FDG PET comparison with nonmutated patients[END_REF]. The conclusion is that C9orf72 mutated ALS patients t have a more widespread central nervous system involvement than ALS patients without genetic mutations, with or without dementia.

A PET study showed a widespread loss of binding of the GABAA ligand [ 11 C]-flumazenil in sporadic ALS patients [START_REF] Lloyd | Extramotor involvement in ALS: PET studies with the GABA(A) ligand [11C]flumazenil[END_REF]. Because GABAA receptors are widely distributed in the cerebral cortex and are located both on pyramidal cells and interneurons, [ 11 C]-flumazenil provides a means of detecting motor and extramotor dysfunction in ALS. It was shown that abnormalities were less extensive in patients with SOD1linked familial ALS patients, suggesting that GABA-ergic neurotransmission may be less severely impaired in these cases [START_REF] Turner | Distinct cerebral lesions in sporadic and 'D90A' SOD1 ALS: studies with [11C]flumazenil PET[END_REF]. Nigrostriatal dysfunction has also been shown by both PET [START_REF] Przedborski | Nigrostriatal dopaminergic function in familial amyotrophic lateral sclerosis patients with and without copper/zinc superoxide dismutase mutations[END_REF] and SPECT studies [START_REF] Borasio | Dopaminergic deficit in amyotrophic lateral sclerosis assessed with [I-123] IPT single photon emission computed tomography[END_REF].

PET imaging can also detect inflammatory processes that are implicated in the pathogenesis of ALS. Assessment of microglial activation can be performed through neuroimaging of the 18 kDA translocator protein (TSPO) that is present in activated glial cells, using selective TSPOselective radioligands such as 11 C-PK11195. Extensive cortical abnormalities in fixation of this tracer, predominantly in the frontal and temporal lobes have been observed in one study [START_REF] Turner | 11C]-WAY100635 PET demonstrates marked 5-HT1A receptor changes in sporadic ALS[END_REF]. The DPA714 radioligand has a longer half-life, a better bioavailability, and less nonspecific binding than PK11195 [START_REF] Chauveau | Comparative evaluation of the translocator protein radioligands 11C-DPA-713, 18F-DPA-714, and 11C-PK11195 in a rat model of acute neuroinflammation[END_REF]. This allows the examination of milder microglial activation by PET. A prospective study showed significant binding of DPA714 both in motor cortex areas and also in temporal areas since the earlier stages of the disease [START_REF] Corcia | Molecular imaging of microglial activation in amyotrophic lateral sclerosis[END_REF]. With the continuing development in new radioligands, PET imaging may potentially provide tools to monitor the effect of drugs targeting inflammation.

Conclusion

In the recent years, thanks to technological and methodological developments, neuroimaging was revealed as an indispensable research tool to understand the pathophysiology of ALS. Because ALS is a multisystem disorder and not a pure motor neuron disease, there is a need to investigate in vivo the participation of other systems such as the sensory or extrapyramidal systems. Neuroimaging studies have already provided insights about the potential role of sensory feedback, inflammation, and loss of inhibition by interneurons in the pathogenesis of ALS. Studies on presymptomatic carriers of mutations responsible for FALS provide the unique opportunity to study the very early mechanism that triggers the neurodegenerative cascade leading to the loss of motor neurons.

Among MRI techniques, although DTI initially appeared as the most promising diagnostic tool, the disappointing results of a meta-analysis suggest that a multiparametric approach will be required to make neuroimaging a critical component in the workup of ALS. Development of spinal cord imaging will be a key element to provide useful biomarkers as sensitive and specific as possible to help diagnosis and to characterize phenotype or progression. Due to the intrinsic heterogeneity of ALS, larger pooled banks of images with standardized image acquisition and analysis procedures are needed. For this purpose, the NeuroImaging Society in ALS (NISALS) has emerged in 2010 and has an interactive web Platform (http://nedigs05.nedig.uni-jena.de/nisals/) to provide quality controlled MRI data for the international scientific community. Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that mainly affects the motor system. At present, the only drug to have produced an increase in patient survival in a controlled clinical trial is riluzole [1]. The condition is always fatal; the median survival time after onset is 36 [2], although there are great individual variations [3]. Although ALS is a clinically recognizable condition (in terms of the pattern of progressive upper and lower motor neuron degeneration), the clinical presentation and progression are heterogeneous [4]. Moreover, there are now strong reasons for considering a broader aetiological and pathogenic spectrum [5]. This clinical heterogeneity is observed in other neurodegenerative diseases, such as Parkinson's disease (PD). Nevertheless, it has been found that all PD patients display degeneration and iron overload of the substantia nigra, which might become a surrogate biomarker [6]. In ALS, iron overload in the motor cortex was confirmed [7] after being suspected in mouse models [8]. Ongoing drug trials in the field of neurodegenerative disease are mainly seeking to establish neuroprotection and neurorestoration. Since neuroprotection can never be unambiguously demonstrated in a given patient, the concept of disease-modifying drugs (with a slowing of the disease progression) has arisen. At present, the evaluation of a drug's clinical efficacy in ALS is based on the ALS Functional Rating Scale Revised, motor tests, survival or a combination of these measures (such as the Combined Assessment of Function and Survival (CAFS)) [9,10]. However, these endpoints are general, variable and late-stage measures of the ALS disease process and thus require the long-term assessment of large cohorts. These assessments are risky and expensive, which considerably limits the number of trials being conducted. A sensitive surrogate biomarker might help to (i) reduce the sample size in pilot studies, (ii) better define the sample size required in Phase III clinical trials (by comparison with clinical scales) and (iii) define endophenotypes for separate assessment in clinical trials.

Biomarkers are typically divided into "wet biomarkers" and "dry biomarkers". Wet biomarkers are biological substances that are measured in a body fluid (such as whole blood, serum, plasma, saliva or cerebrospinal fluid (CSF)). "Dry biomarkers" are based on functional scales, task performance, electrophysiology or imaging. Magnetic resonance imaging is a widely available and non-invasive technique, which makes it an appropriate candidate for biomarkers. Although early reports stated that ALS starts in the spinal cord, the initial site of neurodegeneration has not been unambiguously identified; disease may start in the spinal cord, in the motor cortex or at both sites simultaneously [11,12]. The development of radiological biomarkers in ALS is facilitated by the hypothesis that the pathological changes in ALS radiate out from the initial spinal and brain areas in which the most severe upper and lower motor neuron dysfunction respectively occur (from symptom onset onwards) with variability in the predominance of upper and lower motor neuron dysfunction [13]. The neurodegeneration then progresses along the motor neurons of the ventral horn of the spinal cord and into the corresponding areas of the motor cortex [13]. Despite this modelization, the pattern of onset and progression remains complex [13]. Hence, MRI of the spinal cord should be an early, sensitive marker of subtle degenerationrelated changes. However, MRI of the spinal cord is complicated by a number of technical difficulties (relative to MRI of the brain). Firstly, the magnetic field is inhomogeneous because the spinal cord is close to bone, soft tissues and air. Secondly, the spine has a small cross-sectional dimension, which thus requires high-resolution data acquisition. Thirdly, the spine is subject to motion of the CSF [14] and physiological motion due to breathing [15].

Many different MRI techniques and sequencesincluding voxel-based morphometry (VBM), diffusion tensor imaging (DTI), magnetic resonance spectroscopy (MRS), iron-sensitive sequences (T2*, R2* and SWI) and functional MRI (fMRI) -are available for studying ALSrelated changes in the brain or spinal cord. Hence, we reviewed the correlations between MRI findings and clinical scores in ALS, and sought to establish whether particular sequences may have value as surrogate biomarkers in this disease.

Methods

We searched the PubMed database with the following keywords: "ALS AND MRI OR amyotrophic lateral sclerosis AND magnetic resonance imaging", "ALS AND VBM OR amyotrophic lateral sclerosis AND magnetic resonance imaging ", "ALS AND MR spectroscopy OR amyotrophic lateral sclerosis AND magnetic resonance spectroscopy", "amyotrophic lateral sclerosis AND diffusion tensor imaging OR ALS AND DTI", "amyotrophic lateral sclerosis AND functional magnetic resonance imaging OR ALS AND fMRI". There was no limitation on the publication date but only publications in French or English were considered. We included (i) general MRI studies with a control group and an ALS group and (ii) longitudinal follow-up studies (even if a control group was lacking). We excluded therapeutic trials articles, reviews, studies focused only on particular subgroups of ALS patients such as cognitive impairment, genetic mutations.

Results

A total of 1336 articles were identified using database searching, 651 were recorded after duplicates removal. 405 were excluded (not ALS, not MRI or, therapeutic clinical trials, not English or French, reviews) and 130 articles were finally excluded (studies focused only on particulars subgroups of patients, other MRI sequences or lacking of a control group). We analysed 116 articles.

Brain imaging

Structural magnetic resonance imaging (Table 1)

Three methods based on a three-dimensional, T1weighted sequence have been applied to ALS: VBM, volume analysis and the measurement of cortical thickness. As a volume-based analysis, VBM involves the automatic segmentation of grey matter and inter-subject comparison of the local grey matter density [16,17]. After all the data have been transformed in the same stereotaxic space, the images are partitioned and corrected for the separation between white matter, grey matter and CSF as a function of voxel intensity [17]. Measurement of cortical thickness is a surface-based technique analysis in which the white matter and pial surfaces are automatically extracted by applying image analysis packages such as Freesurfer (http://surfer.nmr.mgh.harvard.edu/).

Most of these techniques have evidenced atrophy in the precentral gyri [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. However, some DTI studies used VBM to show that ALS vs. control differences in the fractional anisotropy (FA) of the precentral gyri were not due to atrophy [33,34]. In other studies, cortical atrophy was not limited to the precentral gyri but extended to other regions of the frontal lobe [18-24, 26-28, 32, 35] -especially in apathetic patients with involvement of the cingulate cortex [27,36]. Other regions involved include several parts of the temporal cortex [19-24, 32, 35], the hippocampus [24,[37][38][39], the parietal cortex (mostly in the post-central cortex [18,21,23,28]) and the insula [24]. Occipital grey matter atrophy [19,20,24,35] and cerebellar atrophy were less common [24]. When considering the basal ganglia, a low volume of the thalamus [22,24,37] and the caudate nucleus [37] have been observed. Few studies have evidenced a relative reduction in the volume of white matter (mainly in the frontal and temporal lobes) [23]. There were no marked differences in the corpus callosum [40]. Whole-brain analyses have evidenced low volumes in the neocortex, grey matter, white matter [28] and brain parenchyma [28,32]. A longitudinal study showed (i) a volume reduction in the left subiculum, the right hippocampal area CA4 and the right dentatus gyrus and (ii) an enlargement of the ventricles in 39 of the 112 ALS patients [38].

Clinical correlations Specific MRI abnormalities have been correlated with clinical scores, the disease stage (as estimated with the original or revised ALS Functional Rating Scale (ALSFRS and ALSFRS-R, respectively), the speed of disease progression (i.e. the change in ALSFRS or ALSFRS-R score, divided by the disease duration or the time interval between two clinical assessments), the disease duration and the survival time. Lower ALSFRS or ALSFRS-R scores were associated with larger ventricles and greater volume loss in the basal ganglia [38], and volume changes in several parts of the frontal lobe (such as Brodman area 10) [18]. In another study, a higher ALSFRS-R progression rate was associated with MRI changes in the left sensorimotor area; cortical thickness was lower in patients with predominantly upper motor neuron impairment (especially in the precentral gyrus and the left paracentral lobule), whereas patients with predominantly lower motor neuron impairment showed less difference relative to controls [20]. Grey matter density was lower in the cingulate and right inferior frontal gyrus in patients with impaired emotional empathy [36]. Poor survival in ALS is correlated with volume loss in the basal ganglia and limbic structures [38]. The initial side of limb weakness in ALS patients was not correlated with contralateral brain cortical loss of volume [27]. Although atrophy was predominant in the left sensorimotor cortex [27,28], it was not related to the side of onset [27]. A subgroup analysis (by side of onset) found more widespread atrophy in the contralateral brain cortex than in the group of ALS patients as a whole [28]. Precentral cortical thickness was lower in patients with upper motor neuron clinical features than in those with lower motor neuron clinical features and in the ALS group as a whole [29].

In a study with a post mortem T1 sequence and histological assessment, an abnormally small difference in intensity between cortical grey matter and subcortical white matter was observed not only in the motor cortex (including the precentral gyrus, supplemental motor area and premotor cortex) but also in somatosensory areas and the primary visual cortex. These signal abnormalities were linked to neuron loss and an elevated number of astrocytes [41].

Diffusion tensor imaging (Table 2) DTI (also known as magnetic resonance tractography) is based on the random diffusion (Brownian motion) of molecules. In a spherical volume, the diffusion of water has no main direction and its diffusion in the three directions (λ1, λ2 and λ3) is equally likely. However, this is no longer the case in an ellipsoid or cylindrical volume and the diffusion is anisotropic. λ1 is the volume's main axis (axial diffusivity, AD) and λ2 and λ3 are the minor axes (radial diffusivity, RD). By analysing the diffusion of water in three directions, four parameters can be defined: FA, RD, AD and mean diffusivity (MD, which is the average of diffusion in the λ1, λ2 and λ3 axis, also referred to as the apparent diffusion coefficient (ADC)). The first three parameters describe the spatial variation of water movement and are related to the orientation of the studied structures. In contrast, MD corresponds to the mean displacement of the water molecules within the volume. The main application of DTI is the analysis of the anatomical bundles that compose the white matter of the brain and the spinal cord [42][43][44]. Whereas DTI corresponds to the analysis of the above-mentioned parameters in a defined volume, tractography corresponds to the analysis of a complete white matter tract.

Fractional anisotropy in ALS Low FA along entire corticospinal tracts (CSTs) is the most common observation in patients with ALS [34,[45][46][47][48][49][50][51][52][53][54][55][56][57][58]. However, some studies have only reported low FA in one or several parts of a CST: the subcortical white matter of the precentral gyrus [23,24,30,33,[59][60][61][62][63][64][65], the corona radiata [24,[60][61][62][66][67][68], the posterior limb of the internal capsule (PLIC) [24,33,60,62,63], the cerebral peduncles [21,59,61] and the pons [61]. Lastly, only one study failed to observe a difference in FA along a CST in ALS patients by region of interest approach whereas tract based spatial statistics (TBSS) showed decreased FA in corona radiate and corpus callosum [68]. Low FA has also been observed in other regions: the frontal lobe (excluding the CST) [21,24,33,34,45,52,56,57,61,64], the cingulum [60], the corpus callosum [21,33,45,50,52,58,61,[67][68][69], the parietal lobe [24,34,45], the temporal lobe [21], parahippocampal areas [61], the hippocampus [34], the insula [34,52], the cerebellum [61] and the thalamus [24,33,53]. In a whole-brain comparison, many other small areas of ALS-modified white matter tracts were observed [70].

A tractography study revealed that the CST volumes of ALS patients are lower than in controls [71]. A DTI study using a tract of interest-based fibre tracking found that the radiological results mirrored the neuropathological staging system [72], suggesting that the disease process extended in the following sequence: the corticospinal tract (stage 1), the corticorubral and corticopontine tracts (stage 2), the corticostriatal tract (stage 3) and the proximal portion of the perforant pathway (stage 4) [73]. The researchers suggested that this MRI-based staging system could be used as a surrogate marker of disease progression in clinical studies of ALS.

Diffusivity in ALS The most common observation in patients with ALS is a relative increase in MD, reflecting changes in white matter tracts. The MD was elevated along entire CSTs [51,53,69] and in parts of CSTs, including subcortical white matter in the precentral gyrus [23,34,58,60,61], the corona radiata [34,60], the PLIC [23,34,60,74], the cerebral peduncles and the pons [34]. Diffusivity was also elevated in several parts of the frontal lobe [23,24,52,58,60,61], the temporal lobe [23,24,52,61], the hippocampal region [24,34,52], the parahippocampal region [60], the parietal lobe [23,58,61], the insula [23,24,52], the cingulum [53,60], the occipital lobe [23], the cerebellum [23,60] (especially in C9ORF72-positive patients) [75], the corpus callosum [23,52,60,69] and the thalamus [53]. Conversely, two studies did not detect any differences in diffusivity in ALS patients vs. controls [21,63].

Radial and axial diffusivity in ALS Several studies have found elevated RD and AD values in the right CST, the right cingulum and the left anterior thalamic radiations (on the basis of changes in FA or MD) [53]. In the PLIC, AD is low [63] and RD is elevated [67] -especially in patients presenting with T2 hyperintensities in CSTs [63]. RD is elevated in white matter connected to the primary motor cortex, the premotor cortex [56] and the corpus Table 1 The main results of volumetric studies (Continued) callosum [56,67,69]. Lastly, RD and AD are elevated in the cerebellum of C9ORF72-positive patients [75].

Longitudinal studies In longitudinal analyses, low FA is observed in the right superior CST [62], precentral subcortical regions, mesencephalic CSTs and parts of cerebellum [61]. FA was found to fall in a progressive, linear manner along the CSTs [34,46,76], and the diffusivity of the external and internal capsule was elevated [61]. AD is elevated in CSTs [77]. However, a recent longitudinal study found that white matter defects (as assessed by DTI) progressed much slowly that in the grey matter defects -notably in the basal ganglia [78].

Clinical correlations Many studies have found that disease progression (as defined by the change over time in the ALSFRS or ALSFRS-R score) is correlated with the changes in FA in CSTs [24,34,45,61,62,77,79]. Furthermore, the disease duration is correlated with FA in entire CSTs or parts of CSTs [24,50,54,56,61,77]. Conversely, the disease duration was negatively correlated with FA in the cerebellum [61], the subcortical white matter of insula, the ventrolateral premotor cortex, the cingulum, the precuneus and the splenium of the corpus callosum [24]. Lower-limb muscle strength was correlated with the contralateral FA in CSTs [60]. Diffusivity values in CSTs and in the parietal lobe, temporal lobe and cerebellum were also correlated with disease progression [52,79]. Diffusivity in CSTs was correlated with disease duration [79], and contralateral diffusivity in CSTs was correlated with spasticity [59].

Whereas both limb-onset patients and bulbar-onset patients differed from controls in terms of FA along CSTs, the two patient groups had similar values [60]. The CSTs were more impaired in bulbar-onset patients than in limb-onset patients when considering FA, MD and RD but not AD [80]. Tractography revealed a large number of differences between controls and patients defined as definite/probable according to the El Escorial criteria, whereas there were fewer differences between controls and a possible/suspected group [79].

Neurophysiological correlations FA in CSTs was related to the central conduction time in a transcranial magnetic stimulation study [33,49] (Iwata et al., [49]; Sach et al., [33]).

Diagnostic accuracy An individual patient data metaanalysis of CST studies concluded that DTI lacks sufficient diagnostic discrimination [81]. The pooled sensitivity and specificity were only 0.68 and 0.73, respectively. Researchers have also suggested that a multimodal approach (combining DTI with methods such as MRS) may be more promising [55].

Magnetic resonance spectroscopy (Table 3) MRS is a non-invasive means of assessing brain metabolism, based on the chemical properties of molecular structures. Results are expressed as peaks. The main brain metabolites monitored with MRS include Nacetyl-aspartate (NAA), choline (Cho, a cell membrane marker), creatine (Cr) and phosphocreatine (Pcr). The Cr + Pcr peak is a marker of energy metabolism; the level of Cr is assumed to be stable and is used to calculate the metabolite ratio. At a field strength of 1.5 T, the peaks for glutamine (Gln), glutamate (Glu, an excitatory neurotransmitter) and gamma aminobutyric acid (GABA, an inhibitory neurotransmitter) overlap to form a complex referred to as "Glx"; higher field strengths are required for more accurate quantification of this metabolites. The simple sugar myo-inositol (Ins) is absent from neurons but present in glial cells. Hence, elevated brain levels of Ins are associated with glial proliferation, whereas low levels are associated with glial destruction [82][83][84].

The main results for the CST concern the ratio between the NAA peak on one hand and the Cho and Cr + Pcr peaks on the other. In ALS patients, low levels of NAA have been observed in the precentral corticosubcortical region [85][86][87][88][89][90][91], in the PLIC [85] and in entire CSTs [55,59,86,92]. Glu and Glu + Gln were found to be elevated in the precentral corticosubcortical region and in the PLIC [85], whereas Cho was found to be elevated in the precentral region [88,93,94] and along the CSTs [47,59,92]. Levels of the inhibitory neurotransmitter GABA were low in the precentral cortex [55,95], and levels of Ins were low in the precentral corticosubcortical region [89].

Whereas the above-mentioned studies used defined voxels for MRS acquisition, whole-brain spectroscopy has revealed low NAA/Cho or NAA/Cr ratios in many other regions [93].

Follow-up studies have shown a decrease in the NAA peak in the precentral cortex (with no differences for other metabolites) [96], a decrease in the NAA/Cho peak and an increase in the Cho/Cr peak [91].

Clinical correlations Patients with definite or probable ALS (according to the El Escorial criteria) had low levels of NAA and high levels of Ins and Cho in the precentral cortex. In patients with possible or suspected ALS, only Cho and (on the left side) Ins were elevated. In another study of suspected ALS patients, only right-side Ins and right-side Cho were elevated [79]. Low NAA and high Cho and Ins levels are associated with clinical disease severity [94]. Lower NAA levels in the precentral gyrus, disease duration and disease progression were intercorrelated [59], and a low NAA/Cho ratio was associated with poor survival [88].

Diagnostic accuracy In a recent study, the combination of MRS and DTI yielded a sensitivity of 0.93 and a specificity of 0.85, whereas the DTI data alone gave values of 0.86 and 0.70, respectively [55]. A criterion combining the MRS data, hypointensity in the precentral gyri and hyperintensity in the CSTs yielded a sensitivity of 0.78 and a specificity of 0.82; when each of the three parameters was considered alone, the sensitivity and specificity values did not exceed 0.71 and 0.75, respectively [90].

Iron imaging

Iron overload appears to be involved in the physiopathology of ALS [8,97]. However, very few studies have analysed the iron content in the brain of ALS patients. A variety of techniques are available, including relaxometry (T2, T2* or R2*) and the susceptibility-weighted imaging [98,99] (SWI, which is probably more sensitive than relaxometry [START_REF] Kearney | Improved MRI quantification of spinal cord atrophy in multiple sclerosis[END_REF]). Multi-echo T2* and R2* sequences are sensitive to magnetic field inhomogeneities. The value of R2* is measured by voxel-by-voxel modelling of the exponential decrease in signal [START_REF] Valsasina | Spatial normalization and regional assessment of cord atrophy: voxel-based analysis of cervical cord 3D T1weighted images[END_REF][START_REF] El Mendili | Validation of a semiautomated spinal cord segmentation method[END_REF]. In postmortem studies, R2* values have been found to be correlated with the iron content [START_REF] Avants | A reproducible evaluation of ANTs similarity metric performance in brain image registration[END_REF].

Susceptibility-weighted imaging and quantitative susceptibility mapping (QSM)

SWI is derived from T2* sequences and can be described as a flow-compensated, high-spatial-resolution T2* weighted sequence. The main advantage over conventional T2* sequence is better contrast [START_REF] Valsasina | Spatial normalization and regional assessment of cord atrophy: voxel-based analysis of cervical cord 3D T1weighted images[END_REF]. QSM is based on the analysis of phase information. The main sources of phase shifts in biological tissues are the iron, calcium, lipid and myelin contents [START_REF] Konrad | Pattern of cortical reorganization in amyotrophic lateral sclerosis: a functional magnetic resonance imaging study[END_REF]. The value of QSM in ALS was confirmed in a post-mortem study, in which the iron content of grey matter was assessed more accurately than that of white matter [START_REF] Tempelmann | Functional motor compensation in amyotrophic lateral sclerosis[END_REF].

The initial studies in the field noted a shorter T2 time in the motor cortex of most ALS patients (vs. controls) [START_REF] Poujois | Brain plasticity in the motor network is correlated with disease progression in amyotrophic lateral sclerosis[END_REF], although this was not confirmed a few years later [START_REF] Greicius | Default-mode network activity distinguishes Alzheimer's disease from healthy aging: evidence from functional MRI[END_REF]. Later studies found a correlation being T2* shortening and iron deposition in the microglia of the motor cortex (according to a post-mortem examination) [7,[START_REF] Poujois | Brain plasticity in the motor network is correlated with disease progression in amyotrophic lateral sclerosis[END_REF][START_REF] Agosta | Sensorimotor functional connectivity changes in amyotrophic lateral sclerosis[END_REF]. Iron overload in the motor cortex was also described in SWI studies [START_REF] Douaud | Integration of structural and functional magnetic resonance imaging in amyotrophic lateral sclerosis[END_REF] and qualitative studies [START_REF] Kearney | Improved MRI quantification of spinal cord atrophy in multiple sclerosis[END_REF]; in the later study, there were no differences between ALS patients and controls in terms of T2 or T2*. A few quantitative studies have looked at white matter or deep grey matter [START_REF] Douaud | Integration of structural and functional magnetic resonance imaging in amyotrophic lateral sclerosis[END_REF][START_REF] Verstraete | Motor network degeneration in amyotrophic lateral sclerosis: a structural and functional connectivity study[END_REF]. There was a trend towards iron accumulation in CST white matter in a relaxometry study [START_REF] Verstraete | Motor network degeneration in amyotrophic lateral sclerosis: a structural and functional connectivity study[END_REF]. Iron overload was also present in the red nucleus, substantia nigra, globus pallidus, putamen [START_REF] Douaud | Integration of structural and functional magnetic resonance imaging in amyotrophic lateral sclerosis[END_REF] and caudate nucleus [START_REF] Verstraete | Motor network degeneration in amyotrophic lateral sclerosis: a structural and functional connectivity study[END_REF]. The use of SWI sequences revealed widespread iron overload and myelin defects in the white matter of all brain lobes and corpus callosum [START_REF] Jelsone-Swain | Reduced interhemispheric functional connectivity in the motor cortex during rest in limb-onset amyotrophic lateral sclerosis[END_REF]. A retrospective study of iron deposition in motor cortex found that quantitative susceptibility mapping was more accurate than T2*, T2 or FLAIR sequences [START_REF] Agosta | Divergent brain network connectivity in amyotrophic lateral sclerosis[END_REF].

A follow-up study has highlighted the progression of a T2 hypointense area in the motor cortex after 6 months, which was strongly and negative correlated with the ALSFRS score [START_REF] Agosta | Sensorimotor functional connectivity changes in amyotrophic lateral sclerosis[END_REF]. Lastly, changes in SWI sequences in the corpus callosum are correlated with the ALSFRS-R score [START_REF] Jelsone-Swain | Reduced interhemispheric functional connectivity in the motor cortex during rest in limb-onset amyotrophic lateral sclerosis[END_REF].

Imaging of the spinal cord

The few studies to have analysed the spinal cord revealed atrophy in the cervical and upper thoracic regions [START_REF] Kollewe | Magnetic resonance imaging in amyotrophic lateral sclerosis[END_REF][START_REF] Luo | Patterns of spontaneous brain activity in amyotrophic lateral sclerosis: a resting-state fMRI study[END_REF][START_REF] Pohl | Proton magnetic resonance spectroscopy of the motor cortex in 70 patients with amyotrophic lateral sclerosis[END_REF]. A DTI study reported low FA, elevated RD and a high magnetization transfer ratio in lateral segments of the cervical spinal cord [START_REF] Luo | Patterns of spontaneous brain activity in amyotrophic lateral sclerosis: a resting-state fMRI study[END_REF]. Atrophy of the cervical and upper thoracic spinal cord progressed over time, whereas the DTI changes were relatively stable [START_REF] Pohl | Proton magnetic resonance spectroscopy of the motor cortex in 70 patients with amyotrophic lateral sclerosis[END_REF]. This observation agrees with the results of a longitudinal brain MRI study showing that grey matter defects (but not white matter defects) progressed over time [78]. Sensory pathways were also involved, with a decreased FA and an elevated MD and elevated RD in the posterior part of the cervical spinal cord [START_REF] Pohl | Proton magnetic resonance spectroscopy of the motor cortex in 70 patients with amyotrophic lateral sclerosis[END_REF][START_REF] Sarchielli | Magnetic resonance imaging and 1H-magnetic resonance spectroscopy in amyotrophic lateral sclerosis[END_REF]. MRS of the cervical spinal cord revealed low NAA/Cr + Pcr and NAA/Ins peaks and an elevated Ins/Cr peak [START_REF] Suhy | Early detection and longitudinal changes in amyotrophic lateral sclerosis by 1H MRSI[END_REF].

Clinical correlations Cervical and upper thoracic spinal cord atrophy was correlated with upper arm muscle strength, as measured by manual testing [START_REF] Luo | Patterns of spontaneous brain activity in amyotrophic lateral sclerosis: a resting-state fMRI study[END_REF]. FA in the cervical spinal cord was correlated with the ALSFRS score. The rate of atrophy in a follow-up study was correlated with the rates of changes of an arm ALSFRS-R score and an arm strength score (in a manual test) [START_REF] Pohl | Proton magnetic resonance spectroscopy of the motor cortex in 70 patients with amyotrophic lateral sclerosis[END_REF]. The NAA/Cr + Pcr and NAA/Ins peaks were positively correlated with the ALSFRS-R score and forced vital capacity but negatively correlated with disease progression [START_REF] Suhy | Early detection and longitudinal changes in amyotrophic lateral sclerosis by 1H MRSI[END_REF].

Functional MRI

Brain fMRI is a recent technique based on blood flow and blood-oxygen-level dependent (BOLD) contrast, which in turn are based on the neurovascular consequences of neuronal activation [START_REF] Gredal | Quantification of brain metabolites in amyotrophic lateral sclerosis by localized proton magnetic resonance spectroscopy[END_REF]. The paramagnetic properties of deoxyhaemoglobin lead to signal reduction in gradient echo sequences [START_REF] Valsasina | Spatial normalization and regional assessment of cord atrophy: voxel-based analysis of cervical cord 3D T1weighted images[END_REF]. Brain fMRI sequences are acquired in the resting state or during performance of a motor, auditory, cognitive, or visual task [START_REF] Pioro | Detection of cortical neuron loss in motor neuron disease by proton magnetic resonance spectroscopic imaging in vivo[END_REF][START_REF] Kalra | Detection of cerebral degeneration in amyotrophic lateral sclerosis using high-field magnetic resonance spectroscopy[END_REF][START_REF] Stagg | Whole-brain magnetic resonance spectroscopic imaging measures are related to disability in ALS[END_REF]. The resting state sequences are quite similar to T2* sequences [START_REF] Valsasina | Spatial normalization and regional assessment of cord atrophy: voxel-based analysis of cervical cord 3D T1weighted images[END_REF].

In the resting state, functional connectivity is based on fluctuations of the BOLD signal. In general, seven "default" networks can be identified: default mode, executive control, visual salience, sensorimotor, dorsal attention and auditory networks [START_REF] Gredal | Quantification of brain metabolites in amyotrophic lateral sclerosis by localized proton magnetic resonance spectroscopy[END_REF][START_REF] Carew | Presymptomatic spinal cord neurometabolic findings in SOD1-positive people at risk for familial ALS[END_REF]. Functional connectivity between specific brain regions and/or networks and the rest of the brain can then be analysed [START_REF] Gredal | Quantification of brain metabolites in amyotrophic lateral sclerosis by localized proton magnetic resonance spectroscopy[END_REF]. Resting-state analysis can also provide information about local activation (by analysing the amplitude of low-frequency fluctuations (ALFF), for example).

The resting state Resting-state studies have evidenced the widespread reorganization of functional connectivity in ALS patients. This reorganization can be observed as either elevated functional connectivity or low functional connectivity. Functional connectivity was found to be low in several parts of the frontal lobe (the right orbitofrontal cortex, the left inferior frontal cortex [START_REF] Zhu | High resolution spectroscopic imaging of GABA at 3 Tesla[END_REF] and especially the motor cortex [START_REF] Foerster | Decreased motor cortexaminobutyric acid in amyotrophic lateral sclerosis[END_REF]) but was elevated in the parietal lobe (the left precuneus and the right angular gyrus [START_REF] Zhu | High resolution spectroscopic imaging of GABA at 3 Tesla[END_REF]) and the left frontoparietal network [START_REF] Zhu | High resolution spectroscopic imaging of GABA at 3 Tesla[END_REF]. This agrees with the elevated sensorimotor connectivity of the left sensorimotor cortex with several regions of the right hemisphere (mainly the cingulate cortex, parahippocampal gyrus and cerebellum crus II) [START_REF] Waragai | Serial MRI and SPECT in amyotrophic lateral sclerosis: a case report[END_REF]. When compared with alterations of the CSTs (according to FA measurements), there were also changes in right sensorimotor connectivity; the connectivity was elevated if the CST was normal in a DTI analysis but was low if the CST was abnormal [START_REF] Waragai | Serial MRI and SPECT in amyotrophic lateral sclerosis: a case report[END_REF]. A network-by-network analysis revealed low activity in the default mode network [START_REF] Waldemar | Focal reductions of cerebral blood flow in amyotrophic lateral sclerosis: a [99mTc]-d,l-HMPAO SPECT study[END_REF] and elevated activity in the sensorimotor network (both of which include parts of the frontal lobe [START_REF] Waldemar | Focal reductions of cerebral blood flow in amyotrophic lateral sclerosis: a [99mTc]-d,l-HMPAO SPECT study[END_REF][START_REF] Abe | Reduced isotope uptake restricted to the motor area in patients with amyotrophic lateral sclerosis[END_REF], although not the same ones). Thalamic connectivity is also elevated [START_REF] Abe | Reduced isotope uptake restricted to the motor area in patients with amyotrophic lateral sclerosis[END_REF].

Resting-state activity (as measured by the ALFF) was elevated in the frontal lobe (the left anterior cingulate, right superior frontal cortex [31] left medial frontal gyrus and right inferior frontal gyrus [START_REF] Kalra | Neuroimaging in amyotrophic lateral sclerosis[END_REF]), the temporal lobe (the right parahippocampal cortex and the left inferior temporal cortex) and the occipital lobe (the middle occipital cortex) [31] but low in the right fusiform gyrus, the visual cortex and right postcentral gyrus [START_REF] Kalra | Neuroimaging in amyotrophic lateral sclerosis[END_REF].

The resting-state connectivity in the frontal cortex is correlated with disease progression and duration [START_REF] Kalra | Neuroimaging in amyotrophic lateral sclerosis[END_REF]. The connectivity between the left sensorimotor cortex and the right parahippocampal gyri and fourth cerebellar lobule is correlated with the ALSFRS-R [START_REF] Waragai | Serial MRI and SPECT in amyotrophic lateral sclerosis: a case report[END_REF]. Connectivity of the dorsal part of the precentral gyri was correlated with hand strength [START_REF] Foerster | Decreased motor cortexaminobutyric acid in amyotrophic lateral sclerosis[END_REF]. Activity in the right parahippocampal gyri was correlated with disease progression, whereas activity in the left anterior cingulate and left temporal gyrus was correlated with cognitive parameters [31].

Activation in fMRI In motor tasks, activation of the motor cortex was elevated [START_REF] Habert | Brain perfusion imaging in amyotrophic lateral sclerosis: extent of cortical changes according to the severity and topography of motor impairment[END_REF][START_REF] Hoffman | Cerebral glucose utilization in motor neuron disease[END_REF][START_REF] Hatazawa | Cortical motor-sensory hypometabolism in amyotrophic lateral sclerosis: a PET study[END_REF] and spread widely across the supplementary motor area, the premotor cortex [START_REF] Kew | Cortical function in amyotrophic lateral sclerosis. A positron emission tomography study[END_REF] and the parietal somatosensory cortex [START_REF] Habert | Brain perfusion imaging in amyotrophic lateral sclerosis: extent of cortical changes according to the severity and topography of motor impairment[END_REF][START_REF] Kew | Cortical function in amyotrophic lateral sclerosis. A positron emission tomography study[END_REF]. Inhibition was low [START_REF] Hoffman | Cerebral glucose utilization in motor neuron disease[END_REF]. Activation of the ipsilateral sensorimotor cortex was possibly linked to a compensatory mechanism in ALS [START_REF] Cistaro | Brain hypermetabolism in amyotrophic lateral sclerosis: a FDG PET study in ALS of spinal and bulbar onset[END_REF]. A visual task was associated with poor activation of the secondary visual cortex and strong activation of associative areas, whereas an auditory task produced delayed activation in the secondary auditory cortex, and somatosensory stimulation produced prolonged activation of the right inferior frontal gyrus and right posterior insular cortex [START_REF] Cistaro | The metabolic signature of C9ORF72-related ALS: FDG PET comparison with nonmutated patients[END_REF]. In a modified "go"/ "no-go" task, activation of the primary motor cortex, other motor areas, the frontal lobe, the insula and the cerebellum was found to decrease over a three-month period [START_REF] Lloyd | Extramotor involvement in ALS: PET studies with the GABA(A) ligand [11C]flumazenil[END_REF]. Using another type of stimulus, activation of the hippocampus also fell after 3 months [START_REF] Lloyd | Extramotor involvement in ALS: PET studies with the GABA(A) ligand [11C]flumazenil[END_REF]. In a theoryof-mind task designed to evaluation the mirror neuron system, ALS patients displayed abnormally strong activation of the right anterior cortical areas [START_REF] Turner | Distinct cerebral lesions in sporadic and 'D90A' SOD1 ALS: studies with [11C]flumazenil PET[END_REF]. A link between the severity of motor weakness and the pattern of activation has never been observed, although the ALSFRS-R score was correlated with the signal change in the sensorimotor cortex in one study [START_REF] Habert | Brain perfusion imaging in amyotrophic lateral sclerosis: extent of cortical changes according to the severity and topography of motor impairment[END_REF]. When considering the clinical phenotype, only a bulbar-onset subgroup showed low activation of the motor cortex during tongue movements [START_REF] Hatazawa | Cortical motor-sensory hypometabolism in amyotrophic lateral sclerosis: a PET study[END_REF]. Shifts in cerebellar or hippocampal activation were correlated with the ALSFRS-R score.

Discussion

When considering the data as a whole, the most diseasesensitive MRI patterns are located in motor regions (and especially along the CST from the cortex to the spinal cord). However, ALS also affects the brain more broadly; other parts of the frontal lobe, the temporal lobe, the hippocampus, the parietal lobe, the cingulum and the insula are commonly involved, whereas the occipital lobe is less frequently involved. For most MRI parameters, patient vs. control differences are mostly apparent in the left (dominant) hemisphere. The various MRI patterns appear to reflect disease severity, progression and duration. With fMRI, abnormally high levels of activation in the motor cortex are probably related to the diseaserelated loss of inhibition. Excessive activation appears to be associated with compensation for the loss of function caused by neuron loss.

Limitations of neuroimaging as a biomarker for ALS

The low average number of participants represents the main limitation: very few published studies have featured more than 30 subjects per group (i.e. ALS and control groups). Together with the differences in the proportions of the various patient phenotypes, this explains the high variability of the results -particularly outside the motor cortex and the CSTs [START_REF] Przedborski | Nigrostriatal dopaminergic function in familial amyotrophic lateral sclerosis patients with and without copper/zinc superoxide dismutase mutations[END_REF].

Only a small proportion of morphometric studies included a whole-brain analysis. The proportion is higher for DTI studies and low (only one study) for MRS. The selection of regions of interest may over-emphasize changes in the CSTs and the motor system and underemphasize changes outside these areas.

Very few studies had a longitudinal design, which is nevertheless required for the full interpretation of abnormalities. The main difficulty in setting up longitudinal studies relates to the rapid disease progression and the appearance of respiratory failure, which prevents patients from lying in the MRI for any length of time. In studies longer than 6 months, the drop-out rate is usually over 50 %. fMRI studies have highlighted both elevated activity and low activity in several parts of the brain. The main physiopathological explanations relate to a physiological compensation response or elevated activity due to the loss of inhibitory mechanisms [START_REF] Abe | Reduced isotope uptake restricted to the motor area in patients with amyotrophic lateral sclerosis[END_REF].

Spinal cord imaging is a rarely applied imaging modality in ALS, despite the pivotal involvement of the anterior horn of the spinal cord in ALS [START_REF] Przedborski | Nigrostriatal dopaminergic function in familial amyotrophic lateral sclerosis patients with and without copper/zinc superoxide dismutase mutations[END_REF]. Spinal cord imaging appears to be most commonly used in studies of multiple sclerosis, since all studies in this disease context found differences (relative to controls) in the cervical spinal cord. To the best of our knowledge, no studies of the whole spinal cord have been publishedperhaps as a result of technical obstacles.

The biomarker concept

Currently, the diagnosis of ALS is based on the revised El Escorial criteria (also known as the Airlie House criteria), which were established in 1998 and published in 2000 [START_REF] Borasio | Dopaminergic deficit in amyotrophic lateral sclerosis assessed with [I-123] IPT single photon emission computed tomography[END_REF]. Although diagnosis during the course of the disease has become relatively easy, the onset of ALS can be masked by clinical overlap with many other diseases [START_REF] Turner | 11C]-WAY100635 PET demonstrates marked 5-HT1A receptor changes in sporadic ALS[END_REF]. The ideal biomarker is not only sensitive and specific (for various disease phenotypes) early in the disease process but must also easy to access [START_REF] Chauveau | Comparative evaluation of the translocator protein radioligands 11C-DPA-713, 18F-DPA-714, and 11C-PK11195 in a rat model of acute neuroinflammation[END_REF]. In ALS, the typical time interval of 12 months between the initial symptoms and disease diagnosis makes this challenge tougher still [START_REF] Chauveau | Comparative evaluation of the translocator protein radioligands 11C-DPA-713, 18F-DPA-714, and 11C-PK11195 in a rat model of acute neuroinflammation[END_REF][START_REF] Corcia | Molecular imaging of microglial activation in amyotrophic lateral sclerosis[END_REF][142]. The best way of determining early biomarkers for ALS may be to monitor cohorts of presymptomatic patients identified by genetic studies [143]. Although wet biomarkers based on blood or CSF analysis are relatively accessible, there are many candidates and study-to-study reproducibility appears to be quite low [5,[START_REF] Chauveau | Comparative evaluation of the translocator protein radioligands 11C-DPA-713, 18F-DPA-714, and 11C-PK11195 in a rat model of acute neuroinflammation[END_REF].

Perspectives

There is currently a need for a robust clinical-radiological description of ALS using MRI as a function of the disease progression, the various endophenotypes and left/right asymmetry (in terms of upper and lower motor neuron impairment, bulbar involvement, and the cognitive profile ranging from frontotemporal dementia, apathy and dysexecutive syndrome to mild attention disorders).

Despite the few number of studies, spinal cord could be a promising region of interest, although this approach is currently limited by technical post-processing difficulties and a sizeable number of large scale and longitudinal studies involving the spinal cord is needed to build stronger MRI biomarkers of the spinal cord. It may be of value to combine several techniques (e.g. atrophy mapping and measurements of the iron content in the grey matter). The results of MRI of the spinal cord are well correlated with the clinical impairment and might be a good surrogate marker.

A comprehensive analysis of MRI parameters in very large cohorts of ALS patients might reveal a radiological surrogate marker for use in clinical trials. Several points should be considered for large scale studies: in 2010, the first Neuroimaging Symposium in ALS (NiALS) had the aim of establishing consensus about the various applications of MRI to the study of ALS and the possibility of multicentre collaboration. Consensus criteria for the main MRI sequences (VBM, DTI, fMRI and spectroscopy) and the main clinical dataset were established [144]. The aim of this consensus was to lead large multicentre and longitudinal studies in imaging research or therapeutic trials [144]. <In our days, studies might better assess the most recent discovers in ALS such as genetic parameters (especially C9ORF72 expansion), the knowledge in cognitive and behavioural impairments even if the patients do not fulfil criteria for frontotemporal dementia [145]. There is still a need for longitudinal studies especially in presymptomatic and early phase of the disease [145]. Following this multicentre aim, a very recent study showed in 253 patients and 189 controls from eight international ALS specialised centre more widespread white matter tract change pooling the analyses than in single-centre analysis and reached estimation of neuropathological changes [146].

Conclusion

The MRI biomarkers appear to be well correlated with disease severity, duration and progression and are more sensitive in the brain and spinal cord motor regions. The built of MRI biomarkers is limited by the clinical various phenotypes but also by the lack of large and longitudinal studies. To date large cohorts with multicentre studies using a standardized MRI protocol are needed.

Figure 1 .

 1 Figure 1. The human motor system ...................................................................................

  Figure 1. The human motor system ...................................................................................

Figure 2 .

 2 Figure 2. The worldwide crude incidence distribution of ALS ...................................................

Figure 3 .

 3 Figure 3. Patterns of motor involvement in different ALS phenotypes .........................................

Figure 4 .

 4 Figure 4. Cervical spine MRI abnormalities in a case of cervical spondylotic myelopathy .................

Figure 5 .

 5 Figure 5. Overview of non-neuromuscular systemic pathology in SMA ......................................

Figure 6 .

 6 Figure 6. A diagnostic algorithm for SMA .........................................................................

Figure 7 .

 7 Figure 7. Spinal cord content, and surrounding tissues and organs ............................................

Figure 8 .

 8 Figure 8. Comparison of 3T and 7T C-spine images .............................................................

Figure 9 .

 9 Figure 9. MRI ghosting artifacts caused by patient motion ......................................................

Figure 10 .

 10 Figure 10. Image distortion in DTI sequence ......................................................................

Figure 11 .

 11 Figure 11. Slices, saturation bands and shim prescriptions for DTI acquisition ..............................

Figure 12 .

 12 Figure 12. Derived indexes from cross-sectional approach .....................................................

Figure 13 .

 13 Figure 13. Angular correlations in SCI patients ...................................................................

Figure 14 .

 14 Figure 14. VBM analysis -Regional distribution of cord atrophy in MS patients ............................

Figure 15 .

 15 Figure 15. TBM analysis -Regional distribution of cord atrophy related to aging ..........................

Figure 16 .

 16 Figure 16. Model of the dynamic of axonal and grey matter degeneration in ALS .........................

  Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16

Figure 1 .

 1 Figure 1. Schematic diagram of the cortical motor system, the CST generated by corticospinal motor neuron (CSMN), descending from the motor cortex, through the internal capsule. Most CST fibers (more than 80% in humans) decussate in the caudal medulla, before descending further through the spinal cord and connecting at the respective height to the alpha motor neuron (AMN). The AMN then projects toward the muscles to form neuromuscular junctions. Modified from Winner et al. (2014).

  Prevalence doubles by a range of 5 years: it reaches 25/100,000 individuals between 60 and 70 years. Prevalence in men is higher than in women but the gender ratio decreases with age (McCombe et al., 2010; Manjaly et al., 2010).

Figure 2 .

 2 Figure 2. Distribution of ALS worldwide: crude incidence. PYFU: person-years of follow-up. From Marin et al. (2016).

Figure 3 .

 3 Figure 3. Patterns of motor involvement in different ALS phenotypes. Red indicates LMN involvement, blue indicates UMN involvement. Darker shading indicates more-severe involvement. From Swinnen and Robberecht (2014).

  lacks sensitivity and specificity to detect abnormalities in ALS, which limits its usefulness in clinical practice. More advanced and quantitative MRI techniques are used in the research setting, enabling investigation of new aspects of the CNS structure and function involvement in ALS (Agosta et al., 2010; Turner et al., 2015).

Figure 4 .

 4 Figure 4. Cervical spine MRI of a 45-year-old woman with cervical spondylotic myelopathy demonstrating extensive spinal cord atrophy and T2 weighted signal change at the C5 and C6 levels despite the absence of frank spinal cord compression. Modified from Ellingson et al. (2015).

Annex A :

 : Neuroimaging to investigate multisystem involvement and provide biomarkers in amyotrophic lateral sclerosis. Annex B: The value of the magnetic resonance imaging as a biomarker in ALS: a systematic review.

Figure 5 .

 5 Figure 5. Overview of non-neuromuscular systemic pathology in SMA. A summary of tissues and organs currently known to be affected in SMA. From Hamilton and Gillingwater (2013).

Figure 6 .

 6 Figure 6. Diagnostic evaluation for SMA. A diagnostic algorithm for SMA and other neuromuscular disorders. EMG, electromyography; NCS, nerve conduction study; RNS, repetitive nerve stimulation; CK, creatine kinase; NMJ, neuromuscular junction; MRI, magnetic resonance imaging; SMARD, SMA with respiratory distress; X-SMA, X-link SMA. Modified from Wang et al. (2007).

Figure 7 .

 7 Figure 7. T2-weighted mid-sagittal (a) and T2 * -weighted axial sections at C2 (b) and T12 (c) vertebral levels showing the spinal cord content, and surrounding tissues and organs. A, Anterior; L, Left; P, Posterior; R, Right; S, Superior; I, Inferior.

Figure 8 .

 8 Figure 8. Comparison of 3T and 7T C-spine images (C5/C6). a: 3T image using the 19-channel commercial coil with 0.5 " 0.5 " 3 mm resolution (acquisition time = 1 min 44 sec). b: 7T image using the 19-channel receive array and four-channel transmit array with 0.5 " 0.5 " 3 mm resolution (acquisition time = 1 min 44 sec). c: 7T image using the 19-channel receive array and 4-channel transmit array with 0.3 mm " 0.3 mm " 3 mm resolution (no interpolation) (acquisition time = 3 min 43 sec). d: 3T image at the mid-vertebra C4 using 64-channel head and neck commercial coil (Siemens 3T Magnetom Prisma), with 0.4 " 0.4 " 6 mm resolution (no interpolation) (repetitions = 3; acquisition time = 5 min 46 sec). Modified from Zhao et al. (2013).

  al., 2005; Noebauer-Huhmann et al., 2007; Fruehwald-Pallamar et al., 2012). A MRI compatible cervical collar could be used to limit involuntary subject motion. The cervical collar enables subjects to lie comfortably in the scanner for a long acquisition time while minimizing artifacts (Yiannakas et al., 2012).

Figure 9 .

 9 Figure 9. MRI ghosting artifacts caused by patient motion. Signal from shoulders and the chest was attenuated using saturation bands.

For

  an exhaustive description of spinal cord MR imaging challenges, the reader is referred to the book by J. Cohen-Adad et al. (Cohen-Adad and Wheeler-Kingshott, 2014) and the review from Stroman et al., Neuroimage 2014.

Figure 10 .

 10 Figure 10. Image distortion in DTI sequence at the interface between the spinal cord, vertebrae and intervertebral disks.

  contrast to clinical context, advanced MRI techniques' acquisition take into account spinal cord imaging constraints, i.e. limiting the influence of motion, partial volume and magnetic field inhomogeneity (Figure 11) (Cohen-Adad et al., 2011), which results in higher resolution, SNR and CNR compared to MR images produced in the clinical setting. Furthermore, advanced MRI techniques are based on mathematical MR signal modeling and processing of individual MR image voxels, and provide, in addition to visual abnormalities identification, quantitative indexes of white matter motor and sensory pathways as well as grey matter layers integrity in the pathological context. Its typical applications are those sensitive to: i) tissue surface, volume or density variations, giving an indirect access to axonal and neuronal loss or atrophy localization, whether global (Cross-sectional area and volume), or local, at the voxel level or its direction (tensor-based morphometry (TBM), voxel-based morphometry (VBM), and surface-based morphometry (SBM)); ii) water movement distribution along and within white matter fibers, giving an indirect access at the voxel level to fiber bundles degeneration, loss and/or their demyelination (DTI, and its extension, high angular resolution diffusion imaging (HARDI), and tractography); iii) white matter macromolecules content, giving an indirect access at the voxel level to fiber bundles myelin loss (magnetization transfer (MT) imaging) (Stroman et al., 2014).

Figure 11 .

 11 Figure 11. Localizer image demonstrating typical patient positioning with minimum lordosis. A: Slice prescription for DTI acquisition, covering C2 to T2 vertebral levels. Saturation bands were set ventrally and dorsally to the spinal cord to limit aliasing and ghosting artifacts. B: Box prescription for manual shimming, ensuring good B0 homogeneity within the imaged portion of the spinal cord. The acquisition was cardiac-gated with slices acquired during the quiescent phase of cardiac-related motion of the spinal cord. From Cohen-Adad et al. (2011).

Figure 12 .

 12 Figure 12. Derived indexes from cross-sectional approach. APW, anterior-posterior width; CE, cord eccentricity; LRW, left-right width.

Figure 13 .

 13 Figure 13. The directional correlations between the spinal cord radius and the motor score (solid), pinprick sensory score (PPSS) (dark grey) and light touch sensory score (LTSS) (light grey). Correlations are plotted for directions with high significance (p < 0.01). The radial axis shows the correlation coefficient and the spinal cord drawing illustrates the main directions in the plot. A, anterior; L, left; P, posterior; R, right. From Lundell et al. (2011).

Figure 14 .

 14 Figure 14. Regional distribution of cervical cord atrophy in different study groups. Sagittal and axial MR images at various cervical cord levels show clusters of significant cord atrophy (color-coded for t values; P < 0.001, uncorrected only for display purposes). (A) Patients with MS versus healthy control subjects (HC). (B) Patients with RR MS versus HC. (C) Patients with SP MS versus those with RR MS. A, anterior, L, left; P, posterior; R, right. Modified from Valsasina et al. (2013).

Figure 15 .

 15 Figure 15. Results of the TBM analysis between young and elderly volunteers (2 sample t-test, p < 0.05, FDR corrected). The red/yellow clusters indicate regions of atrophy in the elderly subjects. From Taso et al. (2015).

SPINE 1

 1 Imaging Project. The primary objective of the SPINE 1 project was to transfer the acquired knowledge on multi-parametric MRI from previous animal and human studies at the brain and upper cervical spinal cord levels to the entire human cervical and thoracic spinal cord[START_REF] Cohen-Adad | Detection of multiple pathways in the spinal cord using q-ball imaging[END_REF][START_REF] Filippi | Magnetization transfer MRI in multiple sclerosis[END_REF][START_REF] Rodegerdts | 3D imaging of the whole spine at 3T compared to 1.5T: initial experiences[END_REF]. The second objective was to capture sensorymotor system involvement in two MND: sporadic ALS and SMN1-linked SMA as well as to quantify the extent of spinal lesions and of the remaining connectivity in traumatic injuries of the spinal cord. The SPINE 1 project included cutting-edge advanced MRI techniques: i) high-resolution anatomic data to visually identify the extent of the spinal lesions and more importantly to infer on white matter and/or grey matter atrophy; ii) high-resolution HARDI and MT imaging techniques to infer on axonal degeneration, loss and demyelination.The objective of my thesis was to optimize and use the multi-parametric MRI approach at the spinal cord level to analyze grey and white matter structures that are impaired in MND, their temporal alterations during the disease course and the functional correlates, as assessed by clinical and electrophysiological examinations. To achieve such an ambitious objective, imaging, clinical and electrophysiological expertise were combined. In this respect, several processing tools were designed, especially those enabling accurate spinal cord atrophy quantification. The developed tools are detailed in Study 1 and 2, and their applications in MND are presented in Study 4 and 5. Supplementary tools, such as DTI motion correction, are directly integrated in our MND studies (Study 3 and 4).

Study 3 -Study 1 -

 31 Electrophysiological and spinal imaging evidence for sensory dysfunction in amyotrophic lateral sclerosis Study 4 -Multi-parametric spinal cord MRI as potential progression marker in amyotrophic lateral sclerosis Study 5 -Cervical spinal cord atrophy profile in adult SMN1-linked SMAMethodological studies Validation of a semi-automated spinal cord segmentation methodBackground and objectivesAtrophy quantification from MR images requires as a first step the separation of the spinal cord from surrounding tissues, i.e. CSF, and to delineate white and grey matter if they were visible on the images. Regions-of-interest delineation can be achieved manually or by using semi-automated or automated segmentation methods. Several segmentation methods with varying degrees of manual intervention, reproducibility and accuracy have been proposed to assess spinal cord atrophy from MR images(Horsfield et al., 2010; Zivadinov et al., 2008; McIntosh and Hamarneh, 2006; Tench et al., 2005; Hickman et al. 2004; Coulon et al. 2002;Losseff et al., 1996). Most published studies have used low-resolution T1-weighted 1.5T images and mainly investigated the upper cervical spinal cord in MS patients. However, validation of cord atrophy as a biomarker in spinal cord diseases requires evaluating these segmentation methods in large segments of the spinal cord using high-resolution anatomical MR images generated from actual 3T MRI systems as well as in diseases other than MS. In this respect, MND are characterized by selective neuronal damage that provides relevant models. Indeed, ALS and SMA are characterized by diffuse degeneration of LMN localized in the anterior horn of all spinal cord levels(Katsuno et al., 2010; Wijesekera and Leigh, 2009). The entire CST also degenerates in ALS(Wijesekera and Leigh, 2009). On the other hand, SCI at the chronic phase offers a model of secondary white matter tract lesions due to Wallerian degeneration(Wyndaele and Wyndaele, 2006).The semi-automated threshold-based segmentation method proposed byLosseff et al. (1996) is one of the most used approaches to quantify atrophy related to its high reproducibility and accuracy.

Fig 1 .

 1 Fig 1. Overview of DTbM steps (S). Slice 1 represents an example of a resampled axial image. Slices 2 and 3 represent the progressive contrast enhancement of slice 1. doi:10.1371/journal.pone.0122224.g001

Fig 2 .

 2 Fig 2. Segmentation and cord straightening. (a) T2-weighted mid-sagittal section in an ALS patient. The yellow plus signs delimit the spinal cord region to be preprocessed and segmented. (b) Mid-sagittal section of the preprocessed data and (c) resulted segmentation mask (orange). (d) Mid-coronal and (e) sagittal sections of the straightened spinal cord. (f) Mid-sagittal and (g) coronal sections of the straightened mask (orange). A, anterior; I, inferior; L, left, P, posterior, R, right, S, superior. doi:10.1371/journal.pone.0122224.g002

Fig 3 .

 3 Fig 3. Cervical spinal cord template and probability tissue map. (a) Coronal (left), sagittal (middle) and axial views (right) of the cervical spinal cord template through mid-vertebral level from C2 to C7. (b) Probability tissue map. Views are arranged in the same order and orientation as in (a). A, anterior; I, inferior, L, left, P, posterior, R, right; S, superior. doi:10.1371/journal.pone.0122224.g003

S1

  Fig. Illustration of the behaviour of DTbM method. Good agreement of DTbM method: (a,b) in a spinal cord region with T2-hypersignal in a SCI patient; (c,d) in an atrophied spinal cord region in an ALS patient; (e,f) in a narrow spinal canal region in an SMA patient. Failure of DTbM method: (g,h) in a region with T2-hypersignal in a SCI patient; (i,j) in an atrophied spinal cord region in an ALS patient; (k,l) in a narrow spinal canal region in an ALS patient. A, anterior; I, inferior, L, left, P, posterior, R, right; S, superior. (TIFF) 31. Meindl T, Wirth S, Weckbach S, Dietrich O, Reiser M, Schoenberg SO, et al. (2009) Magnetic resonance imaging of the cervical spine: comparison of 2D T2-weighted turbo spin echo, 2D T2*weighted gradient-recalled echo and 3D T2-weighted variable flip-angle turbo spin echo sequences. Eur Radiol 19(3): 713-721. doi: 10.1007/s00330-008-1175-7 PMID: 18813933 32. Lichy MP, Wietek BM, Mugler JP, Horger W, Menzel MI, Anastasiadis A, et al. (2005) Magnetic resonance imaging of the body trunk using a single-slab, 3-dimensional, T2-weighted turbo-spin-echo sequence with high sampling efficiency (SPACE) for high spatial resolution imaging: initial clinical experiences. Invest Radiol 40(12): 754-760. PMID: 16304477 33. Tins B, Cassar-Pullicino V, Haddaway M, Nachtrab U (2012) Three-dimensional sampling perfection with application-optimised contrasts using a different flip angle evolutions sequence for routine imaging of the spine: preliminary experience. Br J Radiol 85(1016): e480-489. doi: 10.1259/bjr/25760339 PMID: 22374284 34. Del Grande F, Chhabra A, Carrino JA (2012) Getting the most out of 3 tesla MRI of the spine. Rheumatology Network.

right side in 20 participants and left side in 1 .

 1 Patients with ALS were stimulated on the most affected side (distal hand weakness) or on the dominant side when both sides were equally affected: 17 patients were stimulated on the dominant right side, 1 patient on the dominant left side and 3 patients on the left nondominant but more affected side. Perceptual and motor thresholds (PT and MT, respectively; figure 2) were estimated in both nerves. Stimulus intensity was then adjusted at 9×PT in each subject. A recording surface electrode was stuck in the supraclavicular fossa (Erb's point) ipsilateral to the stimulations with a reference electrode on the contralateral supraclavicular fossa. A needle electrode was inserted in the scalp, 4 cm lateral and 2 cm 2 Iglesias C, et al. BMJ Open 2015;5:e007659. doi:10.1136/bmjopen-2015-007659posterior from Cz, in front of the primary somatosensory cortex (S1) contralateral to the stimulations with a surface electrode for a reference on the ipsilateral ear lobe. Electrophysiological signals were filtered (bandwidth 30-3000 Hz) and amplified (×10 000; D360 8-Channel Patient Amplifier, Digitimer Ltd, Hertfordshire, UK) before being digitally stored (2 kHz sampling rate) on a personal computer for offline analysis (Power 1401 and Signal Software, CED, Cambridge, UK). Each recording session consisted of 200 ulnar nerve and 200 median nerve stimuli alternated randomly (2 Hz). Three recording sessions were performed for each participant.

Figure 1

 1 Figure 1 Spinal diffusion tensor imaging metrics. (A) T2-weighted MRI sagittal slice (rostro (R)-caudal (C) axis) and mean diffusion-weighted images (C6 vertebra level) in one patient with amyotrophic lateral sclerosis (ALS). For each level between C5 and T1 (dashed white lines), the posterior (P, in blue) and left (L) and right (R, in red) region of interests (ROIs) were drawn manually on the mean diffusion-weighted images to include most of the posterior column-medial lemniscus pathway (sensory pathways) and lateral corticospinal tracts (motor pathways), respectively. The posterior ROI was split in two parts for the right and left side of the spinal cord, according to the antero (A)-posterior axis and the slice centre of mass. Box plot charts showing distribution of data for the radial (B) and mean diffusivity (C) in controls (black rectangles) and in patients with ALS (red rectangles): the top and bottom line of the box correspond to the 75th centile (top quartile) and 25th centile (bottom quartile), respectively, and the open circle in the box, to the 50th centile (median). The two bars extend from the 10th centile (bottom centile) to the 90th centile (top centile). The bar caps delimit outliers. The top and bottom filled circles indicate the maximum and minimum values, respectively. The cross within the box indicates the arithmetical mean. *p<0.05.

Figure 2

 2 Figure 2 Perceptual and motor threshold for median and ulnar nerve stimuli. Mean perceptual (A) and motor (B) thresholds for median nerve (left columns) and for ulnar nerve (right columns) estimated in the group of controls (open columns) and of patients with amyotrophic lateral sclerosis (red columns). Vertical bars are ±1 SEM. *p<0.05.

Figure 3

 3 Figure 3 N9 and N20 amplitude. Raw signal (in μV) from recordings at the Erb point level (A and B) and at cortical level (in front of S1, C and D) in one healthy subject (control, A and C; black lines) and in one patient with amyotrophic lateral sclerosis (ALS, B and D; red lines). Dashed lines indicate the 0 level. Upper and lower dotted lines indicate +2×SD and -2×SD levels, respectively. Group data showing the mean amplitude of N9 (left) and of N20 (right) normalised to prestimulus activity in controls (open columns) and in patients with ALS (red columns) after median (E) and ulnar nerve stimulation (F). Vertical bars are±1 SEM. *p<0.05, **p<0.01 and ***p<0.001.

Figure 4

 4 Figure 4 Relationships between diffusion tensor imaging and electrophysiological metrics. N9 (A and C) and N20 (B and D) amplitude (% 2×SD of the prestimulus activity) plotted against the mean diffusivity (×10 -3 mm 2 /s) for median nerve (A and B) and for ulnar nerve (C and D). Each open circle represents one control and each red circle, one patient with amyotrophic lateral sclerosis. Iglesias C, et al. BMJ Open 2015;5:e007659. doi:10.1136/bmjopen-2015-007659

Figure 5

 5 Figure 5 Patient data distribution. Proportion of patients exhibiting normal (white portion) and abnormal diffusion tensor imaging (DTI) metrics and/or abnormal amplitude of N9 (A and B) or N20 (C and D; red portion) for the median (A and B) and ulnar nerve (C and D). Among the patients with abnormal metrics (red portion), the proportion of patients with abnormal DTI metrics only (and thus normal evoked potentials) is illustrated by the blue subportion, the proportion of patients with abnormal evoked potential only (and thus normal DTI) is illustrated by the green subportion, and the proportion of patients with abnormal DTI metrics and abnormal evoked potentials is illustrated by the purple subportion.

Figure 1 .

 1 Figure 1. Regions of interest definition. (A) T2-weighted MRI mid-sagittal slice for an ALS patient. (B,C) T1-weighted data for MTR (B) and mean diffusion-weighted data for DTI (C). (D,E) Defined anatomical ROIs. ROIs were selected in the dorso-lateral (red) aspect of the spinal cord to include most of the CST. (F,G) T2-wheighted data before (F) and after (G) cord outlining. A, anterior, I, inferior; L, left; R, right; P, posterior; S, Superior. doi:10.1371/journal.pone.0095516.g001

Figure 2 .Figure 3 .

 23 Figure 2. Comparisons between ALS patients at baseline and follow-up for individual cross-sectional area and MTR in the dorsolateral aspect of the spinal cord that includes mostly the corticospinal tract. Group differences were assessed using Wilcoxon signed rank test. Levels of significance are indicated as: *P,0.01, **P,0.001. doi:10.1371/journal.pone.0095516.g002

  SMA patients were recruited from the Myology Institute and the Motor Neuron Disease referral Center (Pitié-Salpêtrière Hospital, Paris, France) between May 2009 and March 2011. Local Ethic Committee approved all experimental procedures (Paris-Ile de France Ethical Committee under the 2009-A00291-56 registration number), and written informed consent was obtained from each participant. All clinical investigations and MRI examinations were conducted according to the principles expressed in the Declaration of Helsinki.

Fig 1 .

 1 Fig 1. Spinal cord segmentation, cord straightening and radial distance illustration. (A) Preprocessed T2-weighted mid-sagittal section in an SMA patient. The yellow dashed lines delimit the cervical spinal cord region. (B) Resulted segmentation mask (orange). (C) Cervical spinal cord straightening. (D) Mask straightening (orange). (E) Radial distance measurements. A, anterior; I, inferior; L, left, P, posterior, R, right, S, superior. doi:10.1371/journal.pone.0152439.g001

Fig 2 .Fig 3 .

 23 Fig 2. 3D atrophy profile along the cervical spinal cord in SMA patients. The color-coding indicates regions with significant atrophy (color scale = pvalues of the between groups comparison). A, anterior; I, inferior; L, left, P, posterior, R, right, S, superior. doi:10.1371/journal.pone.0152439.g002

  S1 Dataset. Data set underlying the findings in the present study. MAT-file containing individual MRI data, demographics and clinical features. (MAT) S1 Fig. 3D correlation profile between MMT C5 subscore and RD along the cervical spinal cord. The color-coding indicates regions with p-values < 0.05 (color scale = p-values of the correlation between MMT C5 subscore and RD). (TIFF) S2 Fig. 3D correlation profile between MMT C6 subscore and RD along the cervical spinal cord. The color-coding indicates regions with p-values < 0.05 (color scale = p-values of the correlation between MMT C6 subscore and RD). (TIFF) S3 Fig. 3D correlation profile between MMT C7 subscore and RD along the cervical spinal cord. The color-coding indicates regions with p-values < 0.05 (color scale = p-values of the correlation between MMT C7 subscore and RD). (TIFF) S4 Fig. 3D correlation profile between MMT C8 subscore and RD along the cervical spinal cord. The color-coding indicates regions with p-values < 0.05 (color scale = p-values of the correlation between MMT C8 subscore and RD). (TIFF) S5 Fig. 2D correlation profile between MMT C8 subscore and CSA along the cervical spinal cord. The color-coding indicates regions with p-values < 0.05. For correlations between MMT C5-C7 subscores and CSA along the cervical spinal cord, all p-values were higher than 0.05. (TIFF)

  glial cells and myocytes (Morrison et al. 1998; Ilieva et al., 2009; Chang and Martin 2011; McGown et al. 2013).

Figure 16 .

 16 Figure 16. Putative model of the dynamic of axonal and grey matter degeneration. During the presymptomatic phase, axonal and grey matter lesions progress at slow rate. Close to the onset of clinical signs, grey matter lesions accelerate and play the major role in clinical signs. During the symptomatic period, axonal lesions slowly progress. By contrast, grey matter lesions show rapid progression, possibly in relation to specific propagation mechanisms (i.e. "prion-like"), and functional decline is mainly dependent on the extension of anterior horn cells degeneration. Yellow: CST (motor pathways); blue: grey matter (anterior horn cells); red: dorsal column (sensory pathways).

  Technological and methodological advances in the field of spinal cord MRI bring new perspectives for the clinical setting. Indeed, more and more clinical centers are equipped by 3T MR systems with adapted coil and advanced MRI techniques (DTI, MT imaging, anatomical imaging) to image the spinal cord with high resolution in feasible duration time for patients. Specific processing tools of spinal cord images (De Leener et al., 2016b; Study 1 and 2) will facilitate the transfer from the research setting to clinical practice, which will improve the diagnosis and monitoring of a large range of spinal cord pathologies.

Figure 1 :

 1 Figure 1: Evidence of hippocampal and basal ganglia involvement in amyotrophic lateral sclerosis. Comparative surface-based vertex analyses between healthy controls and C9orf72 hexanucleotide repeat negative ALS patients corrected for age and multiple comparisons reveal significant left hippocampal (a) and thalamic changes (b) (Courtesy of Peter Bede-Trinity College Dublin).

Figure 2 :

 2 Figure 2: (a) T2-weighted turbo spin echo midsagittal section in a healthy subject (male, 62 years old), showing the anatomical landmarks of the cervical spinal cord. (b) T2 * -weighted 2D gradient recalled echo axial sections at the vertebral levels C2, C3, C4, and C5 for the same subject (voxel size = 0.7 × 0.7 × 3 mm). Images have been acquired using a 3 T MRI system (TIM Trio 32-channel, Siemens Healthcare, Erlangen, Germany). A: anterior; I: inferior; L: left; P: posterior; R: right; S: superior.

  Ciccarelli et al.,[45] 26/41 Low along CSTs, the CC, anterior limb of the internal capsule, external capsule, parts of frontal lobe WM and postcentral gyri Correlation between DPR and FA in left cerebral peduncle, right PLIC, right corona radiate right WM adjascent to the precentral gyrus and CC Ding et al., [74] 1 0 / 1 0 E l e v a t e di nt h eP L I C N o ta v a i l a b l e Ellis et al., [51] 22/20 Low along CSTs Elevated along CSTs Correlation between mean diffusivity in CST and DD and between FA along CSTs and ALS severity scale and spasticity scales. Filippini et al., [56] 24/24 Low along CSTs and in the CC Elevated RD in WM linked to the primary motor and premotor cortex and the CC Inverse correlation between FA and UMN score along CSTs, correlation between FA and ALSFRS-R and DD along CSTs Foerster et al., and in the motor part of the CC Elevated along CSTs Correlation between DD and FA along CSTs. Inverse correlation between FA in CSTs and global and localized UMN impairment score and between FA along CST and UMN rapidity index. Kassubek et al., [12, 73] 111/74 Low along CSTs Not available

  Elevated along CSTs, hippocampus, insula, parts of the temporal and frontal lobe and CC Correlation between FA along CSTs, in prefrontal area and ALSFRS. Negative correlation between MD along CST, hippocampus, cerebellum, parietal and temporal lobe and ALSFRS. Sarica et al., [53] 14/14 Low in right CSTs and left anterior thalamic radiations Elevated in right CSTs, cingulum and left anterior thalamic radiations RD elevated in right CSTs and left anterior thalamic radiations. AD elevated in the right cingulum Negative (p ≤ 0.05) Schirimrigt et al., 2007 10/20 Low along CSTs (nb: study with a technical objective) Correlation between FA along CSTs and DD Stagg et al., , in frontal WM and the genu of the CC Elevated in the centrum semi-ovale and frontal and parietal WM Not available Thivard et al., [24] 15/25 Low along CSTs, premotor cortex, right thalamus, insula, parts of parietal lobe Elevated in the motor cortex, premotor cortex, insula, hippocampus, and right superior temporal gyrus Correlation between FA along CST, insula, premotor cortex, cingulum, precuneus, CC and ALSFRS-R, negative correlation between FA in CC and centrum semiovale and DD

Table 1 .

 1 Principal characteristics of ALS phenotypes .............................................................

Table 2 .

 2 The SMN2 gene copy number and the related motor performance in SMA ......................

Table 3 .

 3 The spin density and the relaxation time in some tissues .............................................

Table 4 .

 4 Detected spinal cord MRI abnormalities in "mimic" MND ..........................................

Table 5 .

 5 Correlations between DTI indexes and white matter microstructures... ..........................

	Tableau 1
	Tableau 2
	Tableau 3
	Tableau 4
	Tableau 5

Table 1 .

 1 Principal characteristics of ALS phenotypes from an Italian register of 1332 ALS patients. Modified from Chiò et al.

	(2011).
		Number of cases Age at onset	Median survival
	Phenotype	(%)	Mean ± SD	in years
	Classic	404 (30.3)	62.8 ± 11.3	2.6
	Flail arm	74 (5.5)	62.6 ± 11.8	> 4
	Flail leg	173 (13.0)	65.0 ± 9.6	3
	Bulbar	456 (43.2)	68.8 ± 9.7	2
	Respiratory	14 (1.1)	62.2 ± 8.6	1.5
	Pyramidal	120 (9.1)	58.3 ± 13.5	6.3
	PMA	38 (2.9)	56.2 ± 11.3	7.3
	PLS	53 (4.0)	58.9 ± 10.9	13
	Total	1332	64.3 ± 11.3	-

  Survival probability at 5 and 25 years are 98.5% and 68.5%, respectively. SMA type III, mild-onset Kugelberg-Welander disease, starts after 18 months (12% of total SMA patients). Patients are clinically characterized by a progressive proximal leg weakness more than arms.Patients learn to walk without support but may need a wheelchair at some point of the disease course.Life expectancy is almost normal. The SMA type III group is heterogeneous in terms of maintaining the ability to walk during the course of the disease, thusZerres et al., (1995) proposed a subdivision of this type into two subgroups: IIIa with an onset from 18 months to 3 years, and IIIb with an onset after 3 years. The probability of being ambulatory after 10, 20 and 40 years from the onset is 70.3%, 33.5% and 22%, respectively, in SMA type IIIa; and 96.3%, 84% and 58.7% in SMA type IIIb.

	SMA type III

II, intermediate-onset Dubowitz disease, starts between 6 and 18 months (27% of total SMA). Patients are clinically characterized by a progressive weakness in legs more than in arms, associated with hypotonia and areflexia. Patients are able to sit unassisted but are not able to walk independently.

Table 3 .

 3 The

	Tissue	Relative spin 1.5T		3T
		densities a	T1 (ms)	T2 (ms)	T1 (ms)	T2 (ms)
	Brain
	Grey matter	83	1000	100 (T2), 65 (T2 * )	1331	85 (T2), 42 (T2 * )
	White matter	71	710	80 (T2), 78 (T2 * )	832	70 (T2), 49(T2 * )
	CSF	100	4000	2200	4000	2200
	Spinal cord at C3
	Grey matter	-	-	-	972 b , 994 c	76
	Lateral column	-	-	-	863 b , 830 c	73
	Dorsal column	-	-	-	900 b , 853 c	72
	Skeletal muscle	80	1060	35	1420	32
	Venous blood	82	1435	150	1584	80
	Arterial blood	82	1435	235	1664	165
	Fat	90	300	165	380	133
	a 100 denote the 100% spin density;

relaxation time constants and relative fully relaxed signals, i.e. their spin densities of some tissues. Modified from Atlas SW (2009) and Smith et al. (2008). b inversion recovery sequence; c gradient echo sequence.

Table 4 .

 4 Detected abnormalities from spinal cord MRI in "mimic" MND.

	Involvement site	"Mimic" MND	Spinal cord MRI abnormalities
	Peripheral nerve	Neuropathies	No abnormalities
	Spinal cord	Kennedy Syndrome	No abnormalities
		Hirayama disease	"snake eyes" T2*-hypersignal appearance on
			the cervical anterior horns
		Cervical spondylotic	Narrow spinal canal associated with an
		myelopathy	intramedullary T2-hypersignal
		Hereditary spastic	No abnormalities
		paraplegia

that aging involves grey matter ventral horns neurons loss (Morrison, 1959; Cruz-Sánchez et al., 1998). Thereafter, the same research group used VBM to map the regional distribution of atrophy at the cervical spinal cord level in MS patients with different clinical phenotypes (Valsasina et al., 2013; Rocca et al., 2013) (Figure

  The production of such high quality spinal cord MR images was accompanied

	with adapted and innovative methodological developments to process such images. New image
	processing algorithms using more advanced approaches emerged, and permit exploration of the
	topography of spinal cord atrophy in the pathological context. Such explorations give a new insight into
	the pathophysiological mechanisms involved in spinal cord tissue degeneration. For instance, in our
	previous cross-sectional study in ALS patients (Cohen-Adad et al., 2013) (25 ALS patients versus 20/43
	age-matched controls), we quantified cross-sectional area from high-resolution MR images at the lower
	cervical spinal cord. Our study suggested a specific atrophy profile along the lower cervical spinal cord
	in ALS patients. In addition, we showed that cord atrophy at C7 vertebral level (equivalent to C8 spinal
	level) was correlated with the MEP amplitude obtained after stimulation at the level of C8 nerve root,
	which reflects impairment of lower motor neurons, not CST. Our findings suggested that regional spinal
	cord atrophy as measured using MRI reflects anterior horn cells degeneration in ALS. In MS context,
	one major impact spinal cord MRI study (Klein et al., 2011), explored the susceptibility of the spinal
	cord levels to atrophy in four MS subtypes using volume measurement (two patients with clinically
	isolated syndrome (CIS), 27 with relapsing-remitting MS (RRMS), two with secondary-progressive MS
	(SPMS), and four with primary-progressive MS (PPMS) phenotypes versus age and gender-matched
	healthy controls; the spinal cord was imaged from C1 to T12). Patients with PPMS/SPMS had a
	different atrophy profile compared with RRMS/CIS patients. Patient with PPMS/SPMS showed upper
	cervical cord atrophy. However, RRMS/CIS patients showed an increased volume at the cervical and
	thoracic levels, which most likely mirror inflammation or edema-related cord expansion.
	Advanced atrophy indexes
	Cross-sectional and volume measurements produce global atrophy indexes. Alternatively, some
	studies proposed more advanced and complex methods for a focal cord atrophy quantification, derived
	from volumetric approach. Voxel-based morphometry method was widely used to localize atrophied
	brain regions in the pathological context (O'Callaghan et al., 2016; Tavazzi et al., 2015; Byun et al.,
	2015; Ille et al., 2011). Voxel-based morphometry method is based on statistical analysis of the
	difference in white/grey matter brain tissues concentration between groups. The latter method was
	introduced for the first time to analyze cord atrophy related to aging by Valsasina et al. (2012). Their
	methodological study was a proof of concept of the feasibility of VBM to produce reliable results on
	cervical spinal cord atrophy due to aging (90 healthy subjects). Their study showed that most voxels
	significantly associated with a lower probability of containing cord tissue were located in the ventral

portion of the cord. The latter result is consistent with post-mortem studies, which found

Table 5 .

 5 Pearson's correlations corrected p-values for multiple comparisons (p-values without correction) between DTI indexes and white matter microstructures derived from electron microscopy of a small specimen of the fimbria-fornix. The three other cross-sectional ALS studies, showed a significant change in FA, RD and MD at the cervical spinal cord (p < 0.05)(28 ALS patients versus 20 age-matched controls in Valsassina et al., 2007; 14 ALS patients versus 15 age-matched controls in Nair et al., 2010), and more specifically FA and MD in the CST in ALS patients compared to healthy controls (24 ALS patients versus 16 age-matched controls in Wang et al., 2014). In ALS patients, FA was positively correlated with

	Modified from Concha et al. (2010).
	White matter microstructure	FA	MD	AD	RD
	Axonal density	0.13 (0.0495 * )	0.83 (0.3)	0.66 (0.2)	0.47 (0.12)
	Inner axon diameter	0.64 (0.3)	0.68 (0.21)	0.66 (0.2)	0.74 (0.24)
	Outer axon diameter	0.76 (0.4)	0.83 (0.3)	0.74 (0.24)	0.94 (0.44)
	Myelin thickness	0.11 (0.04)	0.64 (0.19)	0.92 (0.4)	0.29 (0.064)
	Cumulative axon membrane	0.02 (0.007) *	0.52 (0.14)	0.83 (0.3)	0.11 (0.023 * )
	Circumference
	Myelin fraction	0.47 (0.2)	0.83 (0.3)	0.96 (0.47)	0.68 (0.21)
	* P-values > 0.05
	Diffusion tensor imaging was widely used at brain level in MND and almost in ALS, and reported
	change in DTI indexes in parts of the CST as well as outside the motor regions (Turner and Verstraete,
	2015; Agosta et al., 2010). However, at the spine cord level, only a small number of studies used DTI
	to investigate white matter integrity in MND, and they were exclusively in ALS (Cohen-Adad et al.,
	2013; Wang et al., 2014; Nair et al., 2010; Agosta et al., 2009; Valsassina et al., 2007). This was
	mainly related to spinal cord imaging difficulties (see spinal cord imaging challenges), as well as the
	rarity of MND diseases. In our cross-sectional study in ALS patients (Cohen-Adad et al., 2013) (29 ALS
	patients versus 21 age-matched controls), we showed changes in FA and RD in the CST and dorsal
	column (sensory afferents). Fractional anisotropy correlated with ALSFRS-R (p = 0.04) and TMS motor
	threshold (p = 0.02).

Table 2 .

 2 Criteria and scores for the visual evaluation.

	Criteria

2

Insufficient to moderate overlap in less than 3 vertebral levels. 3 Global substantial overlap agreement. 4 Global perfect overlap agreement. doi:10.1371/journal.pone.0122224.t002

Table 1 .

 1 Segmented spinal cord regions for each group of subjects (Upper-Lower vertebral level limits).

	Controls	ALS	SMA	SCI

Table 3 .

 3 Results of the visual evaluation.

	Criteria

*Asymptomatic syrinx revealed by the visual evaluation. doi:10.1371/journal.pone.0122224.t003

the SMA group, the mean DSC value was 95.30% for DTbM, 91.07% for ASM. Finally, in the SCI group, the mean DSC value was 95.75% for DTbM and 91.10% for ASM.

Table 4 .

 4 Accuracy of mean cross-sectional area measured at the cervical and thoracic levels.

	Cervical level	Thoracic level
	Method	Method

Manual segmentation by the experienced operator was the ground truth for accuracy measurements. doi:10.1371/journal.pone.0122224.t004

Table 5 .

 5 Accuracy of mean cross-sectional area measured at C2 vertebral level.

	Method	Method

Manual segmentation by the experienced operator was the ground truth for accuracy measurements. doi:10.1371/journal.pone.0122224.t005

35 .

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Multi-

parametric MRI studies in MND Study 3 -Electrophysiological and spinal imaging evidence for sensory dysfunction in amyotrophic lateral sclerosis Background and objectives

  Animal and human studies in ALS showed evidence of involvement of spinal and cortical interneurones, and structures such as corpus callosum, basal ganglia and the cerebellum(Prell and Grosskreutz, 2013; Thivart et al. 2010; Pradat et al., 2009; van der Graaff et al. 2011; Carr et al. 2010; Özdinler et al., 2011; Shefner and Logigian, 1998). In addition, post-mortem examinations of spinal cords in ALS patients have shown axonal loss within the dorsal roots suggesting an alteration of the sensory afferent pathways at late stages of the disease (Kawamura et al., 1981; Averback and Crocker, 1983;

Bradley et al., 1983

). Peripheral and central degeneration of sensory pathways have also been reported in presymptomatic ALS mice, suggesting that the sensory defect occurs at a very early stage of the degenerative process

[START_REF] Sábado | Accumulation of misfolded SOD1 in dorsal root ganglion degenerating proprioceptive sensory neurons of transgenic mice with amyotrophic lateral sclerosis[END_REF] King et al., 2012; Guo et al., 2009)

. Electrophysiological

  The anatomical damage of dorsal column revealed by spinal DTI was correlated to the depression of the peripheral afferent volley revealed by the lower amplitude of cervicobrachial SEP. All Caroline Iglesias, 1 Sina Sangari, 1 Mohamed-Mounir El Mendili,1 Habib Benali,1 Véronique Marchand-Pauvert, 1 Pierre-François Pradat1,2

		Electrophysiological and spinal imaging
		evidences for sensory dysfunction
		in amyotrophic lateral sclerosis
	To cite: Iglesias C, Sangari S, El Mendili M-M, et al. Electrophysiological and spinal imaging evidences for sensory dysfunction in amyotrophic lateral sclerosis. BMJ Open 2015;5: e007659. doi:10.1136/ bmjopen-2015-007659	ABSTRACT Objectives: The prevalence of sensory impairment at an early stage of amyotrophic lateral sclerosis (ALS) is still debated. The study aim was to investigate the anatomofunctional properties of sensory pathways in patients with ALS, combining spinal diffusion tensor imaging (DTI) and somatosensory evoked potentials (SEPs).
	▸ Prepublication history for
	this paper is available online.
	To view these files please
	visit the journal online
	(http://dx.doi.org/10.1136/
	bmjopen-2015-007659).
	CI and SS are co-first
	authors; VM-P and P-FP are
	co-last authors.
	Received 13 January 2015
	Revised 29 January 2015
	Accepted 2 February 2015
	For numbered affiliations see
	end of article.
	Correspondence to
	Dr Véronique Marchand-
	Pauvert;
	veronique.marchand@upmc.
	fr

together these results strongly suggest that sensory defects have been underestimated in previous studies. Therefore, subclinical sensory impairments can manifest in ALS and should no longer cast doubt on the diagnosis. The defect was correlated to the disease duration but a follow-up study would confirm the progressive sensory affection. Together with our previous MRI study

(Cohen-

Adad et al., 2013), these results demonstrated for the first time that advanced spinal cord MRI techniques are powerful tools to investigate extra-motor system involvement in MND. Design: Case-control study. Settings: ALS referral centre and laboratory of biomedical imaging (Paris, France). Participants: Well-characterised group of 21 patients with ALS with moderate disability (mean amyotrophic lateral sclerosis Functional Rating Scale (ALSFRS) score 39.3±1.0) and no clinical sensory signs and control group of 21 gender and age-matched healthy subjects.

Table 1

 1 Clinical data

	ALSFR-S	Muscular testing

Patient: rank.gender (F, female; M, male).age (years old); Onset: first clinical signs manifested in upper limb (UL) or lower limb (LL) or bulbar (B); Duration of the disease in months; El Escorial criteria for amyotrophic lateral sclerosis (ALS): definite ALS (Definite), clinically probable ALS (Probable), Clinically probable-laboratory supported ALS (Lab-supported); Atrophy observed (+) or not (-) on the side explored during the electrophysiological investigations; ALSFR-S: total score of the revised ALS functional resting scale (Total/maximal score 48) and for handwriting (Hand/4); Muscular testing: in upper and lower limbs and in the neck (Total/150), on the side explored during the electrophysiological investigations (UL/45) and in intrinsic hand muscles (Hand/10).

Iglesias

C, et al. BMJ Open 2015;5:e007659. doi:10.1136/bmjopen-2015-007659

Table 2

 2 N9 and N20 characteristics

		Median		Ulnar
		Controls ALS	Controls ALS
	N9
	Latency	10.7±0.2 10.8±0.2 11.4±0.2 11.3±0.2
	CVp	65.9±0.9 66.6±0.7 62.1±0.9 63.5±1.1
	Amplitude	6.1±0.5	5.1±0.5	5.3±0.8	4.2±0.4
	N20
	Latency	19.0±0.4 19.5±0.3 19.2±0.3 19.6±0.3
	CVc	31.5±1.2 28.3±1.0* 33.4±1.4 30.3±1.2
	Amplitude	7.4±0.2	7.2±0.2	7.4±0.2	7.0±0.2

Table 3

 3 Results of multiple regression analysesRegression: R 2 and p value of multiple regression for N9 and N20 produced by median or ulnar nerve stimuli. Group: effect of the group of participants (amyotrophic lateral sclerosis vs controls), coefficient and p value. λ ⊥ : Effect of the radial diffusivity, coefficient and p value. MD: effect of the mean diffusivity, coefficient and p value. Residuals: coefficient and p value of the constant. *p<0.05, **p<0.01 and ***p<0.001.

		Regression	Group	λ ⊥	MD	Residuals
	Median
	N9	0.27	212.3	2.04×10 +6	-1.96×10 +6	673.4
		0.013*	0.025*	0.05*	0.024*	0.14
	N20	0.13	290.8	1.87×10 +6	-1.21×10 +6	491.4
		<0.05*	<0.014*	0.14	0.24	0.38
	Ulnar
	N9	0.39	136.4	7.51×10 +5	-8.40×10 +5	392.0
		<0.001***	<0.01**	0.1	0.028*	0.06
	N20	0.21	241.8	1.96×10 +6	-1.34×10 +6	629.9
		<0.034*	<0.014*	0.06	0.12	0.18

Table 1 .

 1 Patients demographics and clinical features in the whole population and in the subgroup of patients who had a second MRI (mean 6 SD).

		At baseline		At follow up
	Characteristics	Whole population	Sub-group	Sub-group	Delta	Rate of change
	Gender	9 Females/20 Males	4 Females/10 Males	-	-	-
	Age	53.169.8 years	52.6611 years	53.5611 years	-	-
	Recruitment period	Feb 2010-Feb 2011	Feb 2010-Feb 2011	Jun 2011-Jul 2012	-	-
	Disease duration	26.8626.9 months	28.7627.6 months	39.8628.8 months	11.162.7 months	-
	Site of onset	16 Upper; 11 Lower;	6 Upper; 8 Lower	-	-	-
		2 Upper+Lower
	ALSFRS
	Arm (/8)	5.0062.25	5.7162.30	2.9362.89	248.75%	20.25 units/month
	Leg (/8)	4.7262.47	4.9362.23	3.5062.71	228.99%	20.12 units/month
	Total (/48)	37.1466.35	39.9366.18	30.5769.40	221.47%	20.76 units/month
	MMT
	Arm (/70)	52.90611.90	56.79611.11	45616.44	222.14%	21.06 units/month
	Leg (/70)	59.72617.85	61.14616.94	53.79617.13	212.03%	20.58 units/month
	Total (140)	112.62623.80	117.93618.29	98.00624.95	216.90%	21.64 units/month
	TMS
	Threshold (ms)	68.01620.82	63.65618.46	-	-	-
	Amplitude (mV)	1.9261.38	1.6360.97	-	-	-

doi:10.1371/journal.pone.0095516.t001

Table 2 .

 2 Results of stepwise linear regressions.

		ALSFRS-R			MMT
	Predictors	Arm	Leg	Total	Arm	Leg	Total
	FA	0.7198	0.0003(*)	0.0393(*)	0.7766	0.1280	0.1975
	l H	0.6664	0.4428	0.7222	0.5399	0.1439	0.5993
	Cord area	0.0493(*)	0.1492	0.4142	0.0193(*)	0.0434(*)	0.8317

Dependent variables were the ALSFRS-R (arm, leg and total scores) and the MMT (arm, leg and total scores) at MRI 1 . Predictors were gender, age, disease duration, site of onset, DTI metrics (FA, l H , l // and MD), MTR, CSA, TMS measurements (MEP threshold and amplitude). Significant values are marked with (*). Only significant predictors are represented here. doi:10.1371/journal.pone.0095516.t002

  Wilcoxon signed rank test was performed to evaluate changes in MRI metrics from MRI 1 to MRI 2 . Correlation between changes in MRI metrics over time and progression of clinical disability. Spearman's rank correlation coefficient was used to investigate correlations between MRI metrics and rate of change of clinical disability scores, defined as the change of a variable between MRI 1 and MR 2 scans, divided by the time interval between the two scans.

	Comparisons	between	baseline	and	follow-up
	MRI.

Table 3 .

 3 Results of stepwise linear regressions.Dependent variables were the ALSFRS-R (arm, leg and total scores) one year after MRI 1 . Predictors were gender, age, disease duration, site of onset, DTI metrics (FA, l H , l // and MD), MTR, CSA, TMS measurements (MEP threshold and amplitude). Significant values are marked with (*). Only significant predictors are represented here. doi:10.1371/journal.pone.0095516.t003

		ALSFSR-R
	Predictors	Arm	Leg	Total
	FA	0.5342	0.0022(*)	0.0002(*)
	l H	0.6667	0.1377	0,0093(*)
	Cord area	0.0129(*)	0.7775	0.0140(*)

Table 4 .

 4 MRI metrics at baseline and follow-up in ALS patients (mean 6 SD).

	MRI metrics	Baseline	Follow-up	P-value	Delta	Rate of change
	FA	0.52360.054	0.51260.048	0.168	-	-
	l H (610 23 mm 2 /s)	0.74060.128	0.78261.113	0.127	-	-
	l // (610 23 mm 2 /s)	1.74060.117	1.67960.102	0.052	_	-
	MD (610 23 mm 2 /s)	1.05160.115	1.08760.098	0.068	_	-
	MTR	33.2262.51	30.0063.15	0.0029(*)	29.68%	20.33 units/month
	Cord area (mm 2 )	59.766.7	58.166.9	0.00085(*)	22,73%	20.14 mm 2 /month
	Significant differences are marked with (*).

doi:10.1371/journal.pone.0095516.t004

Table 1 .

 1 Population demographics and clinical features in SMA patients.

	Characteristics	SMA patients	Controls
	Number	18	18
	Age	36 ± 11 years	35 ± 11 years
	Gender M:F	10:8	11:7
	Type	5IIIa, 10IIIb, 3IV	-
	Disease duration	26 ± 15 years	-
	MMT
	Scores
	Arm (/70)	56.3 ± 10.2	-
	Leg (/70)	46.7 ± 15.2	-
	Total (/140)	102.9 ± 24.1	-
	C5 spinal level (/10)	7.3 ± 1.9	-
	C6 spinal level (/10)	8.4 ± 1.9	-
	C7 spinal level (/30)	26.1 ± 4.5	-
	C8 spinal level (/20)	14.4 ± 2.7	-
	Percent changes (%)
	C5 spinal level	73.3 ± 19.4	-
	C6 spinal level	84.4 ± 18.9	-
	C7 spinal level	86.9 ± 15.2	-
	C8 spinal level	72.2 ± 13.5	-
	MFM
	D1 (/39)	19.5 ± 11.1	-
	D2 (/39)	37.4 ± 1.6	-
	D3 (/39)	38.3 ± 1.5	-
	ALSFRS-R
	Arm (/8)	7.1 ± 1.2	-
	Leg (/8)	3.3 ± 1.4	-
	Total (/48)	40.2 ± 3.8	-
	doi:10.1371/journal.pone.0152439.t001

  ; d'Errico et al., 2013; Ling et al., 2010; Kariya et al., 2008; Murray et al., 2008; Kong et al., 2009; Wadman et al., 2012; Boyer et al., 2014).

Table 1

 1 The main results of volumetric studies

	Publication	Numbers of	Main results	Main clinical correlations
		patients/controls
	Abdulla et al.,	58/29	Low volume in the right hippocampus	Verbal memory test performance is correlated
	[39, 80]			with the left hippocampal volume.
	Agosta et al.,	25/18	GM volume reduction in right precentral, left	Negative with ALSFRS-R and DD.
	[21]		inferior frontal cortex and temporal superior cortex.
	Agosta et al.,	44/26	Reduction in cortical thickness in the precentral,	DPR is correlated with the mean cortical thickeness
	[35]		frontal, limbic, parietal, temporal and occipital lobes.	of left sensorimotor cortex.
	Bede et al.,	39/44	GM volume reduction in the thalamus, caudate	Not available.
	[37]		nucleus, hippocampus, left putamen.
	Canu et al.,	23/24	GM volume reduction in the precentral, right	Not available.
	[23]		opercular and angular cortex, WM volume reduction
			in the frontal lobe (especially the subcortical
			motor areas) and the temporal lobe.
	Cerami et al.,	14/20	Low GM density in the anterior cingulate and	Not available.
	[36]		right inferior frontal gyri.
	Chang et al.,	20 (10 ALS and	GM volume reduction in the precentral, frontal and	Not available.
	[22]	10 ALS-FTD)/22	temporal cortex and left posterior thalamus.
	Chapman et al.,	25/22	No intergroup difference in the corpus	Not available.
	[40]		callosum area
	Devine et al.,	30/17	GM volume reduction in the left precentral cortex,	Increased GM volume reduction in the dominant
	[27]		left frontal gyrus, medial frontal gyri and anterior	(left) motor cortex irrespective to the side of limb
			cingulate gyri.	onset. Asymmetric GM reduction of the left
				somatosensory cortex and the temporal gyri in
				in right limb onset ALS
	Ellis et al., [26]	16 (8 bulbar onset	GM volume reduction in the superior, middle and	Compared to limb onset ALS, bulbar onset ALS
		and 8 limb onset)/8	medial frontal cortex. WM volume reduction in the	showed GM volume reduction in the brainstem, the
			right frontal cortex.	cerebellum and in the fusiform gyri (BA 34) and WM
				volume reduction along the left corticospinal tract.
	Grosskreutz et al.,	17/17	GM volume reduction in the precentral, frontal	ALSFRS-R correlated positively with GM volume
	[18]		and parietal cortex. No WM atrophy.	reduction in the right medial frontal gyrus (BA 10).
	Kassubek et al.,	22/22	GM volume reduction in the right primary motor	Negative with ALSFRS-R and DD.
	[32]		cortex and the left medial gyrus and bilateral
			inferior temporal gyrus.
	Meadcroft et al.,	8/6	Post mortem study; loss of signal difference	Not available.
	2014		between WM and GM (mainly in the primary
			motor cortex).
	Mezzapesa et al.,	16/9	GM volume reduction in the precentral, frontal	Not available.
	[19]		and parietal cortex.
	Mezzapesa et al.,	29/20	GM volume reduction in the precentral, frontal,	Different patterns of volume reduction in ALS
	[20]		right temporal and right occipital cortex.	group, high UMN burden, spinal onset and
				faster progression compared to controls.
	Sach et al., [33]	15/12	No difference -study not designed to voxel-based	Not available.
			morphometry.
	Sage et al., [34]	28/26	No difference -study not designed to voxel-based	Not available.
			morphometry.
	Schuster et al.,	93 (60 ALS, 17 with	Correlations between the site of onset and upper	Different patterns of volume reduction in ALS
	[25]	dominant UMN, 16	vs. lower motor neuron involvement, with focal	subgroups: bulbar and spinal UMN signs, only
		with dominant	reductions in cortical thickness in the precentral	spinal UMN signs, classical ALS, UMN ALS-variants,
		LMN)/67	cortex.	LMN ALS-variants or sites of onset compared to
				controls. GM reduction is most important in the
				left precentral cortex in bulbar onset vs. limb onset.
				No correlations of MRI with DPR, DD and ALSFRS-R.
	Thivard et al.,	15/25	GM volume reduction in the precentral, frontal	Not available.
	[24]		and temporal cortex (especially the hippocampus),
			the parietal and occipital cortex, the thalamus and
			the cerebellum. No difference in WM.
	Verstraete et al.,	12/12	GM reduction in the right and left precentral cortex. Not available.
	[30]

Table 2

 2 The main results of DTI studies

	Publication	Numbers of	Fractional anisotropy	Mean diffusivity or apparent	Radial and axial diffusivity	Main clinical correlations
		patients/controls		diffusion coefficient
	Abe et al., [64]	7/11	Low in parts of the right frontal			Not available
			lobe and in the left subcortical
			precentral area
	Agosta et al., [21]	25/18	Low in parts of CSTs, the CC,	No difference		Negative
			parts of the frontal lobe and
			parts of the temporal lobe
	Bede et al., [75]	27/42	Low along CSTs, the CC,		RD was low in the	Not available
			cerebellum, brainstem, occipital		cerebellum, brainstem,
			lobe and opercular and insular		occipital lobe and opercular
			regions		and insular regions
	Canu et al., [23]	23/14	Low in subcortical parts of CSTs	Elevated in parts of the CSTs, left		Correlation between MD in bilateral
			and other parts of the frontal	postcentral gyrus, left insula, parts of the		orbitofrontal region and DD
			lobe	left temporal lobe, right angular cortex,
				parts of the frontal lobe, CC, parts of the
				occipital lobe and parts of the cerebellum
	Chapman et al., [67]	21/21	Low in the CC, corona radiata		RD was low in the CC,	Correlation between FA in parts of CC and
			and PLIC		corona radiate and PLIC. AD	ALSFRS-R and negative correlation with DD,
					was elevated in the left	correlation between RD in parts of CC and
					corona radiata and the	DD
					internal and external
					capsules

Table 2

 2 The main results of DTI studies (Continued)

	Keil et al., [61]	24/24	Low in parts of CSTs,	Elevated in motor areas, parts of CSTs, parts		Correlation between FA along CSTs and
			supplementary motor area, CC,	of the frontal and temporal lobe, and the		ALSFRS-R. Correlation between executives
			parts of the frontal lobe and the	postcentral gyrus		functions (sfs36 score) and FA in cerebel-
			parahippocampal area			lum. At 6 months, negative correlation be-
						tween FA along CSTs and DD correlation
						between FA along CSTs and frontal WM
						and ALSFRS-R. Correlation between FA in
						CST at brainstem level and executives func-
						tions, negative correlation between ADC in
						the cerebellum and parahippocampal gyri
						and executives functions (sfs36)
	Keller et al., [68]	33/30	Low in the corona radiata and			Negative
			CC
	Liu et al., 2014	19/13	Low along CSTs			Correlation between FA along left CST and
						ALSFRS-R
	Menke et al., [77]	21/0 (follow-up	Progressive reduction in the			Correlation between FA at baseline and DPR
		study)	PLIC
	Metwalli et al., [69]	1 2 / 1 9	L o wa l o n gC S T s ,t h eC C	E l e v a t e da l o n gC S T s ,t h eC C	A Da n dR Dw e r ee l e v a t e d	Negative
					along CSTs, RD was
					elevated in the CC, parts of
					frontal and parietal lobe
	Muller et al., 2011	19/19	Low in parts of CSTs, the			Correlation between FA in parts of CSTs
			parahippocampal area, insula			and ALSFRS-R
			and brainstem
	Nickerson et al., [46]	2 / 0( f o l l o w -u p	A linear reduction along CSTs			Not available
		study)	during one year follow-up
	Poujois et al., [65]	19/21	Low in CSTs from the left			Muscular strength is lower on the right side
			corona radiate to the precentral			corresponding to the lower FA in the left
			gyrus and in both cerebral			CST
			peduncles
	Prell et al., [60]	17/17	Low in parts of CSTs and the	Elevated parts of CSTs, parts of frontal lobe,		Correlation between FA in internal capsule
			cingulate gyrus	cingulate gyrus, parahippocampal region,		and contralateral strength of the lower limb.
				CC, cerebellum.		Different patterns in FA and ADC of bulbar
						and limb onset compared to controls.
	Prudlo et al., [70]	22/21	Low throughout the CSTs, the			Correlation between FA in many voxels of a
			anterior limb of the internal			whole DTI brain analyses with ALSFRS-R
			capsule, thalamic radiations, the
			CC, association fibres and the
			middle cerebellar peduncle
	Pyra et al., [59]	14/14	Low in left precentral gyrus			Correlation between spasticity and ADC in
						contralateral precentral gyrus
	Rajagopalan et al., [63]	47/10	Low in the left subcortical motor		AD was low in the PLIC. RD	Not available
			area and right PLIC		was elevated in the PLIC of
					patients with T2
					hyperintensities in the CSTs

Table 2

 2 The main results of DTI studies (Continued)

	Rosskopf et al., [57]	100/93	Low along CSTs

  FA fractional anisotropy, MD mean diffusivity, ADC apparent diffusion coefficient, RD radial diffusivity, AD axial diffusivity, CST corticospinal tract, PLIC posterior limb of the internal capsule, CC corpus callosum, DD disease duration, DPR disease progression rate, UMN upper motor neuron, LMN lower motor neuron

	Verstraete et al., [30]	12/12	Low in the rostral part of CSTs		Not available
			and the CC
	Wang et al., [71]	16/17	Low CST volume in DTI		Negative
	Yin et al., [48]	8/12	Low along CSTs		Not available
	Zhang et al., [62]	17/19	Low in parts of CSTs	Elevated in parts of the CSTs	Correlation between FA in right superior
					CST and ALSFRS-R and motor subscore of
					ALSFRS-R

Table 3

 3 main results of magnetic resonance spectroscopy studies Cho and ins were elevated in the MC.NAA and Cr were correlated in left MC Correlation between ins in MC and UMN disability, negative correlation between Naa in MC and UMN disability, higher Cho in sever UMN disability group and Cho was elevated in most parts of CST, and Cho/NAA was elevated in all parts of the CSTs Negative correlation between Cho/Naa in the left entire CST and forced vital capacity, negative correlation between Cho/Naa in the left CST and right and left finger tap rate, negative correlation between Cho/Naa in left MC and semiovale centrum and right finger tape or forced vital capacity Han et al., [85] 15/15 NAA/Cr peak was low in the MC and PLIC, Glu/Cr and Glu + Gln/Cr peaks were low in the MC and PLIC Negative correlation between Glu + Gln/Cr with Norris score Kalra et al., [88, 89] 63/18 NAA/Cho and NAA/Cr was low in the MC, and Cho/ Cr was elevated in the MC Relation between decreased Naa/Cho in the MC and reduced survival. Kalra et al., [88, 89] 17/15 NAA/Ins, NAA/Cr and NAA/Cho were low in the MC, and Ins/Cr was elevated in the MC Negative (p ≤ 0.05) Liu et al., [87] 19/13 NAA/Cr was low in the MC Negative Lombardo et al., [79] 32/19 NAA/Cr was low and ins/Cr and Cho/Cr were elevated in the MC. Progressive decrease over time in the NAA peak throughout the MC Progressive decrease of Naa/(Cr + Cho) in the less affected hemisphere. Correlation between Naa and the more or less affected side (ALSFRS subscore). Correlation between Naa/(Cr + Cho) and the less affected side (ALSFRS subscore) Verma et al., [93] 21/10 NAA/Cho was low in the right lingual gyrus, parts of the occipital lobe, left supramarginal gyrus and left caudate. NAA/Cr was low in the right MC, left frontal inferior operculum, right cuneus, parts of the occipital lobe, left caudate and left Heschl gyrus Not available MC motor cortex, CST corticospinal tract, PLIC posterior limb of the internal capsule, WM white matter, NAA N-acetylaspartate, Cho choline, Gln glutamine, Glu glutamate, PCr phosphocreatine, Cr creatine or creatine + Pcr (the distinction is not relevant for interpretation), DD disease duration, DPR disease progression rate

	Publication	Numbers of	Main results	Main clinical correlations
		patients/controls
	Bowen et al., [94]	18/12
	Cervo et al., [90]	84/28	NAA/(Cho + Cr) was low in the MC	Negative
	Foerster et al., [95]	10/9	Low gamma aminobutyric acid in the MC but not in	Negative
			WM
	Foerster et al., [55]	29/30	NAA and gamma aminobutyric acid were low and	Negative correlation between gamma aminobutyric
			ins was elevated in the left MC	acid in MC and DD, correlation between Naa peak in
				MC and ALSFRS-R
	Govind et al., [92]	38/70	NAA was low Abnormalities were correlated with the El Escorial
				score
	Pohl et al., [91]	70/48	NAA, Pcr + Cr, NAA/Cho and NAA/(Pcr + Cr) were	Not available
			low in the MC. At 12 months, NAA/Cho was low and
			Cho/(Pcr + Cr) was elevated
	Pyra et al., [59]	14/14	NAA/Cho and NAA/Cr were low in the MC and	Correlation between Naa/Cho peak in MC and DD,
			corona radiata, and Cho/Cr was elevated in the MC	negative correlation between Naa/Cho in MC and
				corona radiata and DPR
	Rooney et al., [86]	10/9	NAA/(Cho + Cr) was low in the MC and CST but not	Correlation between Naa/(Cho + Cr) in the MC and
			in other regions	maximum finger tape rate
	Stagg et al., [47]	13/14	NAA was low along the CSTs	Correlation between Naa peak along CSTs and
				ALSFRS-R
	Unrath et al., [96]	8/0

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April 2014 | Volume 9 | Issue 4 | e95516

PLOS ONE | DOI:10.1371/journal.pone.0152439April 18, 2016

(a) (b)

Remerciements

Acknowledgments

We are grateful to all subjects and their relatives. We thank Kevin Nigaud, Romain Valabrègue, Alexandre Vignaud, Frédéric Humbert, Christelle Macia, Mélanie Didier and Eric Bardinet from CENIR for helping with the acquisitions. We thank Drs Mark A. Horsfield for providing us the trial version of Jim 6.0. We thank the clinicians of the Paris ALS Center, Gaëlle Bruneteau, Lucette Lacomblez, Marie-Violaine Lebouteux, Timothée Lenglet, François Salachas and Vincent Meininger. We thank the clinicians of Pitié-Salpêtrière Hospital, Drs Anthony Behin, Tanya Stojkovic, Pascal Laforêt and Bruno Eymard for their contribution. We thank also Thierry Albert from Coubert Hospital, Caroline Hugeron from Raymond-Poincaré Hospital, Robert Lafaye de Micheaux and Isabelle Petit from Invalides National Institute for their contribution.

Acknowledgements The authors thank Dr N Leforestier, Dr T Lenglet, Dr F Salachas, Dr G Bruneteau and Dr V Meininger for their help in patient recruitment, and Dr R Morizot-Koutlidis for her help in the experimental design for SEP recordings. They also thank C Macia, K Nigaud, F Humbert and R Valabrègue for their valuable technical assistance during MRI acquisitions, which were performed at CENIR-ICM Hôpital Pitié-Salpêtrière. Their gratitude also goes to S Blancho for her help to obtain the approval of the local ethics committee and for monitoring the project. At last, they deeply thank Dr Anna Hudson (Neuroscience Research Australia) for scrutinising the English.

Contributors VM-P and P-FP were involved in conception and design of research and P-FP in patient selection. CI, SS and VM-P were responsible for acquisition and analysis of electrophysiological data, CI and M-MEM for MRI acquisitions, and M-MEM for DTI analysis. CI, SS, M-MEM and VM-P was responsible for statistical analysis; HB for supervision of MRI and statistical analysis; CI, SS, M-MEM, VM-P and P-FP for interpretation of the data; and CI, M-MEM and VM-P for drafting of the manuscript and figures. All authors have approved the final version of the manuscript.

Acknowledgments

We are grateful to all subjects and their relatives. We thank Kevin Nigaud, Romain Valabre `gue, Alexandre Vignaud, Fre ´de ´ric Humbert, Christelle Macia and Eric Bardinet for helping with the acquisition and Dr. Henrik Lundell for providing us the code to measure the cord area. We thank the clinicians of the Paris ALS Center, Drs Gae ¨lle Bruneteau, Lucette Lacomblez, Marie-Violaine Lebouteux, Timothee Lenglet and Franc ¸ois Salachas.

Acknowledgments

We are grateful to all subjects for their participation to the study. We thank Kevin Nigaud, Romain Valabrègue, Frédéric Humbert, Antoine Burgos, Mélanie Didier and Eric Bardinet from CENIR for helping with the acquisitions.

Acknowledgments

The authors thank the French Association for Myopathies (AFM), the Institute for Research on Spinal Cord and Brain (IRME), and IHU-A-ICM via the program "Investissements d'avenir" (ANR-10-IAIHU-06) for their research support to the cited studies performed by the authors of the paper.

Acknowledgments

The authors thank Dr. David Fraser (Biotech Communication, Damery, France) for editing the article.

Funding This work was supported by IRME (ID RCB 2012-A00016-37) and ANR (ANR-12-JSV4-0007-01). CI was supported by a grant from FRM, M-MElM by IHU-A-ICM Pitié-Salpêtrière (ANR-10-IAIHU-06), and SS by a grant from UPMC.

Funding Not applicable.

Availability of data and materials

Data and the findings are fully available without restriction upon request to the authors.

S1 Table. Results of the individual visual scoring. (XLSX)

Ethics approval The study conformed to the standards set by the latest revision of the Declaration of Helsinki and has been approved by the ethics committee of Pitie-Salpetriere Hospital (CPP-Ile-de-France VI).

Provenance and peer review Not commissioned; externally peer reviewed.

Data sharing statement No additional data are available.

Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http:// creativecommons.org/licenses/by-nc/4.0/

Author Contributions

Results synthesis

Manual muscle testing revealed a diffuse weakness in the upper limbs involving the muscles innervated by the C5 to C8 spinal segments. Cross-sectional area measurements revealed a significant cord atrophy gradient mainly located between C3 and C6 vertebral levels with a cord atrophy rate ranging from 5.4% to 23% in SMA patients compared to controls. Radial distance was significantly lower in SMA patients compared to controls in the anterior-posterior direction with a maximum along C4 and C5 vertebral levels. There were no correlations between atrophy measurements, strength and disability scores. No correlation was detected between rostro-caudal cross-sectional area gradient and proximal-distal muscles strength variation. As showed by radial distance and cross-sectional area comparisons between SMA and controls, atrophy predominates in the spinal segments innervating the proximal muscles. However, there was a clear distal weakness and no correlations between MRI atrophy measurements and MMT were detected. Our findings suggest that beyond motor neuron loss, other factors such as neuromuscular junction or intrinsic skeletal muscle defects may play a role in the complex mechanisms underlying weakness in SMA.

Erratum: In the "Clinical examination" sub-section, C7 (wrist flexion/extension) should be replaced by C7 (wrist flexion/extension and elbow extension).

Author Contributions

Discussion

Methodological developments

Multi-parametric studies in MND

Perspectives

Methodological developments

In our first methodological study, we validate the semi-automated segmentation method from Losseff's et al. (1996), which represents one of the most used approaches to quantify atrophy at 1.5T.

The method was validated at 3T in the cervical and thoracic spinal cord in a large set of healthy volunteers and three groups of patients with ALS, SMA and SCI. The method showed high reproducibility and accuracy for measuring spinal cord cross-sectional area in both cervical and thoracic regions at 3T. Improved image spatial resolution obtained at 3T and data preprocessing allowed higher segmentation performances than reported previously (Losseff et al., 1996, McIntosh et al., 2011;[START_REF] Horsfield | Rapid semi-automatic segmentation of the spinal cord from magnetic resonance images: application in multiple sclerosis[END_REF]Hickman et al., 2004). However, a limitation of our study is that the method is based on contrast difference between the CSF and the spinal cord, which potentially limit its use in regions with T2-hypersignal at the CSF/SC boundary, e.g. demyelination lesions, and narrow spinal canal, e.g. cervical spondylotic myelopathy and SCI. In that respect, we proposed additional preprocessing steps to circumvent the method limitations as well as to increase its accuracy, i.e. by correcting for partial volume and segmentation imperfections (Horsfield et , 1991). Another limitation of our study is that Losseff's method manual is time-consuming due to its semi-automated characteristic (mean ± SD computational time = 8.0±1.0 min/subject), which restrains its use to investigate atrophy in large portions of the spinal cord in feasible time (Cohen-Adad et al., 2011, 2013), and ultimately its transfer in clinical setting. Hence, the method automation is needed.

Our previous study suggested the possible full automation of Losseff's method, given its low sensitivity to the initialization steps. This led us to conduct a new study with the aim to design a fast and accurate semi-automated segmentation method for spinal cord 3T MR images, benefitting from the high robustness of Losseff's method to its initialization procedure. The computation time for DTbM was

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Abstract

Background: Amyotrophic lateral sclerosis (ALS) is a fatal, rapidly progressive neurodegenerative disease that mainly affects the motor system. A number of potentially neuroprotective and neurorestorative disease-modifying drugs are currently in clinical development. At present, the evaluation of a drug's clinical efficacy in ALS is based on the ALS Functional Rating Scale Revised, motor tests and survival. However, these endpoints are general, variable and late-stage measures of the ALS disease process and thus require the long-term assessment of large cohorts. Hence, there is a need for more sensitive radiological biomarkers. Various sequences for magnetic resonance imaging (MRI) of the brain and spinal cord have may have value as surrogate biomarkers for use in future clinical trials. Here, we review the MRI findings in ALS, their clinical correlations, and their limitations and potential role as biomarkers. Methods: The PubMed database was screened to identify studies using MRI in ALS. We included general MRI studies with a control group and an ALS group and longitudinal studies even if a control group was lacking. Results: A total of 116 studies were analysed with MRI data and clinical correlations. The most disease-sensitive MRI patterns are in motor regions but the brain is more broadly affected. Conclusion: Despite the existing MRI biomarkers, there is a need for large cohorts with long term MRI and clinical follow-up. MRI assessment could be improved by standardized MRI protocols with multicentre studies.

Competing interests

The authors declare that they have no competing interests about this manuscript.

Consent for publication

Not applicable as this manuscript is a systematic review and not a clinical trial.

Ethics approval and consent to participate

Not applicable as this manuscript is a systematic review and not a clinical trial.