This dissertation would not have been possible without many people who have inspired, challenged, supported, and trusted me. To each of them, I would like to express my appreciation and deepest gratitude. I am incredibly fortunate to have Pr Bruno Stankoff as my advisor. I would like to thank him for shaping my approach to neuroimaging research and for sharing with me his unique insights on MS pathophysiology. His guidance and patience, never condescending and always supportive, have allowed me to not only explore the world of research but to blossom as a scientist.

I don't think I would be where I am today if it were not for Pr Catherine Lubetzki. Her passion for the cause of MS and the compassion she shows her patients and students have inspired me tremendously. I can't thank her enough for her support throughout my career and for the opportunities she has opened for me.

I am very grateful to Pr Jerry Wolinsky, who has followed up with me since my first observership in his department and later welcomed me to his lab as a post-doctoral fellow. I am truly indebted to him for taking the time to share with me his clinical and research expertise and for encouraging my independence.

I would like to thank Dr Michel Bottlaender for teaching me about PET imaging and for his critical input.

My sincere thanks also go to Pr Gilles Edan, Pr Jeroen Geurts, Pr Aurélie Kas and Dr Françoise Durand-Dubief for accepting to serve on my thesis review committee. I look forward to your challenging questions, your critique of this work and your valuable insights on future endeavors. I would like to thank my colleagues for their contribution to this work and for their words of encouragement, particularly Celine Louapre, Benedetta Bodini, Daniel Garcia-Lorenzo, Laure Bottin, Claire Leroy, and Benjamin Granger. This work would not have been possible without the neurologists of the MS team, Dr Rana Assouad, Dr Caroline Papeix, and Dr Elisabeth Maillart, the nurses of the Centre

Introduction 13

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system (CNS), affecting about 2 million individuals worldwide. The disease remains a major public health issue as the leading cause of non-traumatic neurological disability among young adults. Significant progress has been made in the past decades to address the inflammatory component of the disease, which has lead to the emergence of more successful disease modifying therapies (DMT) and to better clinical outcomes for patients at least in the early phases of the disease. Yet our picture of MS remains fuzzy at best, and our reach in terms of therapy limited as none of the drugs currently available has been shown to successfully prevent disability progression.

The classical dichotomy between inflammation and neurodegeneration had to be challenged to further our understanding of progression in MS. While inflammatory and demyelinating lesions of the white matter (WM) remain the hallmark of the disease and the most characteristic marker for diagnosis, grey matter (GM) changes and in particular neuronal damage is now thought to play a major role in the neurological decline of MS patients. Recognizing that besides myelin, synapses, dendrites and neurons are early targets of the disease process could have profound therapeutic implications. While this has been suggested in post-mortem tissue samples, further evidence in patients at different stages of the disease is critically needed. Several have tried to measure neurodegeneration in vivo using brain volumetry or non-conventional magnetic resonance imaging (MRI). But these techniques for the most part lack specificity to the neuronal compartment. Thus the development of a new imaging technique able to specifically assess the neuronal component of GM damage in MS patients may have significant impact.

GENERAL CONSIDERATIONS

Since its first pictorial descriptions by Jean Cruveilhier in his Anatomie Pathologique du corps humain [1] and Robert Carswell in Pathological Anatomy: Illustrations of the Elementary Forms of Disease [2], followed by its identification and classification as a distinct entity by Jean Martin Charcot, we have come a long way in our ability to diagnose and document the features of la sclérose en plaques. Multiple Sclerosis is now recognized as one of the most common diseases of the central nervous system (CNS),

and is a very active field of research.

Epidemiology

In a recent review of existing literature, Melcon et al. [3] estimated the median MS incidence at 5.2 (range 0.5-20.6) per 100.000 p-yrs and median prevalence at 112/100.000 pop. Incidence and prevalence were found highest in North America.

While low in childhood, MS incidence peaks between 20 and 40 years of age with a mean age of onset of symptoms often found around 30 years [4].

What was thought as a predominantly male disease at the beginning of the 20 th century is now known to affect women disproportionately. Sex ratios of about 2-2.5/1 have been frequently reported but could be as high as 4.1 in some US communities [5]. The sex prevalence of MS seems to undergo dynamic changes, with several recent examples of increasing sex ratios. In Denmark, Koch-Henriksen and colleagues reported that the incidence of MS in men had remained virtually the same whereas that of women had doubled since 1970 [6]. This phenomenon may indicate potential environmental changes, not just a better diagnosis of benign cases in women.

MS prevalence varies greatly between continents. As mentioned above, North America was found to have the highest incidence and prevalence in a recent study [3], followed by Europe. In contrast, South and East Asia, South America and North Africa had much lower rates. However the notion of a latitudinal gradient suggested by early epidemiologic studies is now being increasingly questioned as genetic and environmental factors are thought to play a key role over geography in the prevalence and incidence of MS. Of particular interest, lower circulating levels of vitamin D have been associated with a higher risk of MS [7] [8]. It has also been shown that vitamin D levels are inversely associated with clinical relapses and MRI activity in MS patients [9].

Other environmental factors such as cigarette smoking [10] and Epstein-Barr virus infection [11] have been identified.

Diagnosis of Multiple Sclerosis

Clinical features

Initial presentation can greatly vary from patient to patient. Common presenting symptoms include: optic neuritis, brainstem or spinal cord manifestations or in less frequent instances hemispheric symptomatology. In up to one fourth of cases, symptoms at presentation may be multifocal [12]. When a patient presents with symptoms suggestive of WM tract damage, the exclusion of an alternate diagnosis is imperative before a diagnosis of MS can be made. Such diagnosis will then rely on the demonstration of "dissemination in space" (DIS), and "dissemination in time" (DIT)

based on clinical grounds alone (clinically definite MS or CDMS) or a combination of clinical and radiological findings.

A "relapse" is defined as "patient-reported symptoms or objectively observed signs typical of acute inflammatory demyelinating event in the CNS […] with duration of at least 24 hours, in the absence of fever or infection" [13]. Based on the McDonald criteria of the International Panel on Diagnosis of MS, initially published in 2001 [START_REF] Mcdonald | Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis[END_REF],

subsequently revised in 2005 [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF] and 2010 [13], a diagnosis of MS can be reached on clinical findings alone if the patient presents with a history of 2 or more relapses and objective clinical evidence of 2 or more lesions. It should be expected for MRI findings to be consistent with a diagnosis of MS, though not mandatory in this case. In all other presentations (2 attacks with objective evidence of only one lesion, single relapse, progressive course), MRI will play a central role in demonstration of DIS and DIT.

Magnetic Resonance Imaging

MRI is currently the most useful paraclinical tool for the diagnosis of MS. MS white matter plaques, the pathological hallmark of the disease, can be detected with great sensitivity particularly on T2-weighted or Fluid attenuated inversion recovery (FLAIR)

sequences. Their objective presence on MRI is considered an essential requirement for the diagnosis of MS. These lesions are often periventricular with a characteristic ovoid shape, but can also be seen in juxtacortical or infratentorial areas (Figure 1). MRI lesions enhancing after injection of Gadolinium (Figure 2) reflect active inflammation and breakdown of the blood-brain barrier (BBB) and are thus considered more recent (≤3 weeks on average). MRI in CIS patients, as it seems to have limited value [START_REF] Rovira | Evidence-based guidelines: MAGNIMS consensus guidelines on the use of MRI in multiple sclerosis-clinical implementation in the diagnostic process[END_REF]. However, it is important to perform spinal MRI for differential diagnosis in patients presenting with symptoms suggestive of spinal cord demyelination [START_REF] Kearney | Spinal cord MRI in multiple sclerosis--diagnostic, prognostic and clinical value[END_REF]. Compressive myelopathy can easily be ruled out. Other disorders can present with spinal cord lesions and the topography and extent of the lesions should be critically analyzed. Particular consideration will need to be given to neuromyelitis optica (NMO). In MS, short-segment lesions (figure 3) are the rule, whereas NMO lesions are longitudinally expansive (more than three vertebral segments in length) on sagittal views. MS lesions are often located on the periphery of the cord on axial views. They contrast with NMO lesions, which are often central and expand axially within the spinal GM. While earlier diagnostic criteria using MRI were based on lesion number [START_REF] Barkhof | Comparison of MRI criteria at first presentation to predict conversion to clinically definite multiple sclerosis[END_REF], revised and simplified criteria by Swanton and colleagues now focus on lesion location (periventricular, juxtacortical, infratentorial, spinal cord) for demonstration of DIS [START_REF] Swanton | Modification of MRI criteria for multiple sclerosis in patients with clinically isolated syndromes[END_REF]. With widespread availability of MRI, we have seen an increase in incidental abnormal findings. Radiologically isolated syndromes (RIS) are defined as MRI findings suggestive of MS in persons without typical MS symptoms and with normal neurological findings. This entity was recently introduced in the phenotypic description by Lublin et al. [START_REF] Lublin | Defining the clinical course of multiple sclerosis: the 2013 revisions[END_REF]. RIS individuals often have brain MRI performed for reasons such as headaches [START_REF] Granberg | Radiologically isolated syndrome--incidental magnetic resonance imaging findings suggestive of multiple sclerosis, a systematic review[END_REF]. Okuda et al. [23] proposed a formal definition of RIS. The RIS Consortium recently presented the results of a retrospective study of 451 RIS subjects from 22 databases in 5 countries [START_REF] Okuda | Radiologically isolated syndrome: 5-year risk for an initial clinical event[END_REF]. This study showed that 34% of RIS individuals would develop an initial clinical event within 5 years of RIS diagnosis. Age < 37 years, male sex, and spinal cord involvement appear to be the most important independent predictors of symptom onset. Prospective follow-up of RIS subjects should shed more light on this entity and help define the basis for treatment with disease modifying therapies (DMTs).

Confirmation

Summary of 2010 revision of McDonald criteria

Prognosis and prediction

It is important to understand clinical phenotypes as a dynamic process. Patient with CIS may convert to RRMS, and patients with RRMS may subsequently follow a SP course.

Brownlee et al. [START_REF] Brownlee | Earlier and more frequent diagnosis of multiple sclerosis using the McDonald criteria[END_REF] young age [START_REF] Mowry | Clinical predictors of early second event in patients with clinically isolated syndrome[END_REF], presence of cognitive impairment at onset [START_REF] Feuillet | Early cognitive impairment in patients with clinically isolated syndrome suggestive of multiple sclerosis[END_REF], genetic factors such as HLA-DRB1 [START_REF] Zhang | Relationship between HLA-DRB1 polymorphism and susceptibility or resistance to multiple sclerosis in Caucasians: a meta-analysis of non-family-based studies[END_REF], and vitamin D deficiency [8]. One of the most significant predictors of conversion to MS from CIS is the presence of brain abnormalities on baseline MRI [START_REF] Tintore | Baseline MRI predicts future attacks and disability in clinically isolated syndromes[END_REF],

with number, location and activity of the lesions all providing prognostic information.

While relapses may have unforgiving consequences in rare cases, the socio-economic impact of the disease is driven by the increasing and unrelenting disability progression that can follow the RR phase. Scalfari et al. recently provided a review of the London Ontario MS database [START_REF] Scalfari | Onset of secondary progressive phase and long-term evolution of multiple sclerosis[END_REF], which evaluated 806 patients annually or semi-annually for 28 years (shortest follow-up=16 years). None of the patients received DMTs. At the end of the study period, 66.3% of patients had developed a SP course. The authors demonstrated that the rate of conversion to SPMS increases proportionally to disease duration. However they highlighted the fact that individual prognosis was highly variable. About 25% of patients will become progressive within 5 years of onset of the disease, while on the opposite end of the spectrum, 25% of patients will remain RR at 15

years. This natural history study confirmed previous findings suggesting that male sex [START_REF] Vukusic | Prognostic factors for progression of disability in the secondary progressive phase of multiple sclerosis[END_REF] and older age of onset [START_REF] Stankoff | Age at onset determines the occurrence of the progressive phase of multiple sclerosis[END_REF] were significant risk factors for conversion to SPMS. The role of early clinical activity in the probability and latency of secondary progression remains debated. Annual relapse rates are today the primary endpoint of many controlled clinical trials, and some have argued it could be used as a surrogate for disability progression [START_REF] Sormani | Surrogate endpoints for EDSS worsening in multiple sclerosis. A meta-analytic approach[END_REF]. Total relapse numbers were however found to have no or little significant effect on the risk of progression, the latency to onset of the SP phase or attainment of high disability levels [34] [35].

Beyond conversion to SP course, physical disability in the clinical setting or in research trials can be assessed using the Expanded Disease Severity Scale (EDSS, Annex A). This scale quantifies disability in eight functional systems (FS). These ratings are then used in conjunction with observations and information concerning gait and use of assistive devices to rate the EDSS from 0 (normal neurological examination) to 10 (death due to MS). In a recent publication [START_REF] Tintore | Defining high, medium and low impact prognostic factors for developing multiple sclerosis[END_REF] may not be influenced by relapses either before or after onset of the SP phase [START_REF] Confavreux | Early clinical predictors and progression of irreversible disability in multiple sclerosis: an amnesic process[END_REF]. Leray and colleagues [START_REF] Leray | Evidence for a two-stage disability progression in multiple sclerosis[END_REF] proposed the concept of MS as a 2-stage disease. The early phase is defined from clinical onset to irreversible EDSS= 3.0 and is thought to be mainly dependent on focal damage to the WM. The second or late phase, from irreversible EDSS=3.0 to irreversible EDSS=6.0, is thought to be independent of focal inflammation and may instead be underpinned by diffuse inflammatory and neurodegenerative changes. In an observational study of 2054 patients referred to the Rennes MS clinic, the authors were able to show that disability progression in the first phase of MS does not influence progression during the second phase. Duration of the early phase was found to be highly variable while the duration of the late phase was remarkably constant, as illustrated in figure 5. The dissociation between the 2 phases of MS, with different underlying mechanisms, is supported by MRI evidence. Calabrese et al.

followed RRMS patients with at least 5 years of clinical history (mean>10 years) and showed that at this stage, variables such as T2 lesion volume did not influence the risk of entering the secondary progressive phase of the disease [START_REF] Calabrese | The changing clinical course of multiple sclerosis: a matter of gray matter[END_REF]. Patient prognostication is a complex and dynamic task in MS, as different clinical, radiological and biological factors can influence outcome depending on the disease stage. This reality reflects the variety of pathological mechanisms thought to underpin disability progression in MS, which will be discussed in a later section

Disease-modifying therapies available in 2015

The last 20 years have seen the emergence and systematic use of DMT in MS. There are currently 13 FDA-approved therapies with many more emerging drugs under trial or consideration. Available DMTs primarily target the inflammatory process of the disease and their mechanisms of action are illustrated in figure 4.

This major development in the MS therapeutic arsenal has almost exclusively benefitted patients with RRMS. All currently approved therapies have been shown to prevent, with varying degrees of efficacy, exacerbations and new brain T2 and Gadolinium enhancing lesions in RRMS. When studied in CIS, first line injectable immunomodulators such as glatiramer acetate (GA) or interferons (IFN) also reduced the risk of conversion into clinically definite MS [START_REF] Castro-Borrero | Current and emerging therapies in multiple sclerosis: a systematic review[END_REF].

The notion of "sustained disability progression", as used in RRMS clinical trial outcomes is defined as a worsening of EDSS performance that persists for 3 to 6 months. As noted by Lublin et al. [START_REF] Lublin | Defining the clinical course of multiple sclerosis: the 2013 revisions[END_REF], worsening in RRMS can occur in the context of multiple attacks, poor recovery after severe exacerbation or true onset of a progressive phase. Kalincik et al., recently suggested that disability outcomes of clinical trials based on 3-6 months "confirmed disability progression" overestimate the accumulation of permanent disability by 30% [START_REF] Kalincik | Defining reliable disability outcomes in multiple sclerosis[END_REF]. Consequently reported effects on "sustained disability progression" may not reflect actual efficacy on the accrual of disability occuring independently of relapse activity. In fact, when tested in SP or PPMS, none of the currently available therapies have demonstrated any effect in slowing progression or reversing disability, leaving about half of the MS patient population with a sense of neglect and frustration.

It has become clear that the pathological processes driving the progressive phase of the disease differ from that of the RR phase. To develop future therapies able to slow, prevent or even reverse progression, we will need to understand the specificity of this disease process, especially the early phenomena contributing to the accumulation of disability. We will need to develop reliable biomarkers that can substitute for clinical endpoints to speed up drug development. Such biomarkers may also allow early identification of patients at risk of progression.

MECHANISMS OF DISABILITY PROGRESSION

White matter damage

Focal demyelination and inflammation

The pathological hallmark of the disease is the formation of WM plaques of demyelination. Clinical relapses are considered the physical expression of these WM lesions. In the early stages of the disease, active WM tissue demyelination within plaques is associated with significant inflammation, BBB damage and microglial activation. Inflammatory infiltrates composed of clonally activated CD8+ T-lymphocytes and to a lesser degree CD4+ T-lymphocytes and B-cells are characteristically detected around post-capillary venules or scattered throughout the brain parechyma. Data suggest that the extent of T-and B-cell infiltration correlates with the degree of demyelination in focal active lesions [START_REF] Babbe | Clonal expansions of CD8(+) T cells dominate the T cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction[END_REF]. Remyelination can occur in MS lesions. This process is particularly stable and extensive in animal models of the disease, but more limited in a majority of MS patients. A study of 168 WM lesions showed that only 22%

were completely remyelinated as "shadow plaques", 73% partially remyelinated and 5%

completely demyelinated [START_REF] Patani | Remyelination can be extensive in multiple sclerosis despite a long disease course[END_REF].

Axonal transection, also a prominent feature of acute and active lesions, appears related to local inflammatory changes [45] [46]. Several factors may contribute to axonal transection during acute inflammatory injury of the WM. Activated T-cells may initiate a pro-inflammatory cascade resulting in the production of interferon gamma, which will subsequently activate macrophages to produce nitric oxide (NO). NO is a potent mitochondrial inhibitor and will disrupt ATP synthesis. Excitotoxicity due to increased release of glutamate by microglial cells or macrophages during the inflammatory process may further hinder mitochondrial function. Glutamate release leads to overstimulation of glutamate receptors on the post-synaptic membrane of neurons and loss of calcium homeostasis. Increased intracellular calcium concentrations activate enzymes involved in cytoskeleton disruption, DNA damage and mitochondrial dysfunction, leading to acute loss of axonal integrity [START_REF] Su | Axonal degeneration in multiple sclerosis: the mitochondrial hypothesis[END_REF].

Focal damage to the WM can be particularly well appreciated using MRI. T2-weighted sequences can detect WM plaques with great sensitivity. Beyond the diagnostic process, MRI can help detect subclinical disease activity by the presence of contrast-enhancing lesions or presence of new/enlarging lesions on subsequent scans. These markers of disease activity are particularly useful to the clinician to evaluate response to therapies which currently target the inflammatory process of the disease. Treatment non responders, or suboptimal responders, in whom a change of therapy should be considered, can thus be identified [START_REF] Rovira | Evidence-based guidelines: MAGNIMS consensus guidelines on the use of MRI in multiple sclerosis-clinical implementation in the diagnostic process[END_REF]. However, as we have seen earlier, MRI studies fail to show significant correlations between focal WM demyelination and inflammation and severity of progression, especially once the patient reaches a certain threshold of disability [48] [49] [39] [38].

To this day, conventional MRI cannot differentiate WM lesions that are fully or partly remyelinated from fully demyelinated ones, though this may be of importance in our understanding of the impact of WM lesions. Indeed, experimental studies have established that remyelination may promote short-term neuronal function recovery and help prevent subsequent axonal degeneration, possibly via trophic effects of axonmyelin interactions [START_REF] Franklin | Neuroprotection and repair in multiple sclerosis[END_REF]. At the patient population level, studies of post-mortem tissue have shown diversity in the amount of remyelination between MS cases independently of the disease stage [START_REF] Patrikios | Remyelination is extensive in a subset of multiple sclerosis patients[END_REF]. A patient may thus exhibit extensive or low remyelinating capacity. This was further confirmed by PET study of MS patients using radiotracer [ 11 C]PIB, a thioflavine derivative sensitive to changes in myelin content in tissue (figure 7) [START_REF] Stankoff | Imaging central nervous system myelin by positron emission tomography in multiple sclerosis using [methyl-(1)(1)C]-2-(4'methylaminophenyl)-6-hydroxybenzothiazole[END_REF]. We recently longitudinal follow-up of MS patients with [ 11 C]PIB PET to support the notion of a patient-specific "remyelination profile". We demonstrated that patients' dynamic remyelination potential was strongly associated with clinical scores (Bodini et al., manuscript in revision), bringing novel insights on the role of WM lesions in MS pathophysiology.

Diffuse white matter damage

In the progressive phase of the disease, inflammation becomes much less pronounced within plaques. Overall the percentage of an individual's lesions that are active declines as the disease evolves [START_REF] Frischer | Clinical and pathological insights into the dynamic nature of the white matter multiple sclerosis plaque[END_REF]. Lesions are either inactive or slowly expanding at the edges.

Similarly, BBB leakage is much less evident [START_REF] Frischer | The relation between inflammation and neurodegeneration in multiple sclerosis brains[END_REF]. This results in a decrease in frequency of contrast-enhanced lesions and new/expanding T2 lesions on MRI, which become poor markers of evolution at this stage.

A characteristic feature of progressive MS is diffuse pathology of brain tissue, outside of focal lesions. Abnormalities have been described in the so-called "normal-appearing WM" (NAWM), ie WM tissue that appears normal both on macroscopic examination and MRI [START_REF] Mahad | Pathological mechanisms in progressive multiple sclerosis[END_REF]. Pathologically, almost 75% of macroscopically normal white matter samples have been found histologically abnormal [START_REF] Allen | A histological, histochemical and biochemical study of the macroscopically normal white matter in multiple sclerosis[END_REF]. Areas of "dirty-appearing WM"

(DAWM) have also been characterized on MRI as having an intensity higher than that of the NAWM, but lower than focal lesions. DAWM can be found in direct proximity of focal lesions or in locations not related to WM lesions and may represent a separate pathologic entity [START_REF] Seewann | Diffusely abnormal white matter in chronic multiple sclerosis: imaging and histopathologic analysis[END_REF].

Pathology of the normal appearing white matter (NAWM) has been well described by post-mortem studies and has been found to differ significantly from that of early focal WM plaques. Kutzelnigg et al. analyzed 52 post-mortem MS brains at different stages of the disease and provided a clear picture of the diffuse pathology affecting the WM [START_REF] Kutzelnigg | Cortical demyelination and diffuse white matter injury in multiple sclerosis[END_REF] (figure 8). The authors reported a global reduction in myelin staining in the NAWM due to decreased fiber density as well as diffuse axonal injury, which is most significant in the progressive MS cases compared to RRMS or acute MS. Axonal pathology is evident by the presence of axonal swellings, axonal end-bulbs and degenerating axons throughout the WM. It may be of importance to note that axonal degeneration is increased around MS lesions and in tracts emerging from plaques. Inflammation is also diffuse at the later stages of the disease and consists of perivascular cuffs of mononuclear cells and diffuse tissue infiltration by T-lymphocytes. Scattered microglial activation is another significant component of NAWM pathology and is profound at the later stages of the disease. Activated microglia, while present diffusely in the NAWM, tend to aggregate in the periplaque of slowly expanding, also called smoldering, lesions where it is associated with ongoing axonal injury [START_REF] Frischer | Clinical and pathological insights into the dynamic nature of the white matter multiple sclerosis plaque[END_REF]. Microglial cells are the resident macrophages of the CNS and can be activated following tissue injury [START_REF] Ciccarelli | Pathogenesis of multiple sclerosis: insights from molecular and metabolic imaging[END_REF]. They can then adopt diverse phenotypes, either protective or driving the degenerative process of the disease. Studies in animal models of MS have suggested that blood-borne monocytes, morphologically indistinguishable from resident microglia, may also infiltrate the brain parenchyma and play an important role in potentiating axonal degeneration [START_REF] Ajami | Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool[END_REF].

Finally, meningeal inflammation, present at all stages of the disease, may influence the extent/topography of diffuse white matter damage. reported in a larger number of subjects significant reduction of myelin density, extensive axonal loss and chronic gliosis [START_REF] Seewann | Diffusely abnormal white matter in chronic multiple sclerosis: imaging and histopathologic analysis[END_REF]. Interestingly these changes appeared chronic in nature as no evidence of acute axonal damage, remyelination or BBB leakage were found.

Several mechanisms may have a role in the extensive axonal damage reported outside of MS lesions. The findings observed in the DAWM support a secondary neurodegenerative process as a result of distant inflammatory lesional damage (wallerian degeneration) [START_REF] Seewann | Diffusely abnormal white matter in chronic multiple sclerosis: imaging and histopathologic analysis[END_REF]. Chronic demyelination in these areas may also imply loss of myelin trophic support, as suggested by evidence of axonal degeneration associated with genetic ablation of myelinating oligodendrocytes in animal models [START_REF] Franklin | Neuroprotection and repair in multiple sclerosis[END_REF]. Loss of myelin staining in the NAWM however appears related to loss of fiber rather than primary demyelination. Recent studies in animal models have suggested that oligodendroglia may support axonal integrity through myelin-independent mechanisms. Impairment of these mechanisms may induce axonal damage without considerable demyelination. In particular, transport of lactate mediated by MCT-1 appears crucial to maintain axon function and survival [START_REF] Lee | Oligodendroglia metabolically support axons and contribute to neurodegeneration[END_REF]. Such axo-oligodendrocyte coupling may be particularly relevant when axons are energy-deprived [START_REF] Funfschilling | Glycolytic oligodendrocytes maintain myelin and longterm axonal integrity[END_REF].

By definition, NAWM is difficult to characterize using conventional MRI techniques such as T1-or T2-weighted sequences. MRI studies have used non-conventional imaging methods to assess the chonology of diffuse WM changes and the impact these changes can have on disease and disability progression. These advanced techniques include diffusion tensor imaging (DTI), magnetization transfer imaging (MTI) or Proton Magnetic Resonance Spectroscopy ( 1 H-MRS). Quantitative MRI studies have shown that diffuse WM changes are present from the early stages of the disease [START_REF] Asaf | Injury to white matter tracts in relapsingremitting multiple sclerosis: A possible therapeutic window within the first 5 years from onset using diffusion-tensor imaging tract-based spatial statistics[END_REF] and increase significantly over time. MRI studies have supported post-mortem evidence of NAWM damage occuring independently from focal damage [58] [65]. DAWM quantitative MRI measures, as reported by Vrenken et al., are intermediate between values of the NAWM and that of focal lesions [START_REF] Vrenken | Diffusely abnormal white matter in progressive multiple sclerosis: in vivo quantitative MR imaging characterization and comparison between disease types[END_REF].

MTI is a technique that is sensitive to microstructural changes in MS and its main metric, magnetization transfer ratio (MTR) is affected by myelin content and possibly axonal loss in post-mortem tissue [START_REF] Schmierer | Magnetization transfer ratio and myelin in postmortem multiple sclerosis brain[END_REF]. Liu et al. found not only a significant decrease in MTR in the NAWM of MS patients compared to controls, but the authors were able to detect a clearly greater disease effect close to the ventricles [START_REF] Liu | Magnetization transfer ratio measures in normal-appearing white matter show periventricular gradient abnormalities in multiple sclerosis[END_REF]. It had already been

shown that MS lesions are also more likely to occur around the ventricles [START_REF] Narayanan | Imaging of axonal damage in multiple sclerosis: spatial distribution of magnetic resonance imaging lesions[END_REF]. Several factors could account for this pattern. Diffusion of CSF-mediated factors related to levels of meningeal inflammation has been suggested. CSF from MS patients has been shown

to impair oligodendrocyte progenitor cells [START_REF] Haines | Multiple sclerosis patient-derived CSF induces transcriptional changes in proliferating oligodendrocyte progenitors[END_REF] as well as neuro-axonal function [START_REF] Vidaurre | Cerebrospinal fluid ceramides from patients with multiple sclerosis impair neuronal bioenergetics[END_REF].

Decreased oxygen availability could also account for these findings as periventricular white matter is particularly prone to hypoperfusion in MS [START_REF] Narayana | Hypoperfusion and T1-hypointense lesions in white matter in multiple sclerosis[END_REF].

Metabolic neuro-axonal function can be assessed in the MS WM by evaluating N-acetylaspartate levels using Proton Magnetic Resonance Spectroscopy ( 1 H-MRS). The extent of NAA reduction within the NAWM at the early stages of the disease was found to correlate with subsequent cognitive deficits [START_REF] Zaaraoui | Occurrence of neuronal dysfunction during the first 5 years of multiple sclerosis is associated with cognitive deterioration[END_REF]. These results echo similar findings using MTI [START_REF] Deloire | Cognitive impairment as marker of diffuse brain abnormalities in early relapsing remitting multiple sclerosis[END_REF] and support the concept of a disconnection syndrome as an underlying mechanism of cognitive disability in MS.

PET imaging, using new tracers able to detect activated microglia and macrophages in vivo, has the potential to better our understanding of the role of diffuse microglial infiltration in disability progression. The translocator protein receptor (TSPO), a protein complex expressed by the outer mitochondrial membrane, is upregulated in activated microglial cells and macrophages, while maintaining low levels of expression in the brain parenchyma. These properties have encouraged the use of TSPO as a target for PET tracers. Compounds such as [ 11 C]PK11195, [ 11 C]PBR28 [START_REF] Park | C-PBR28 imaging in multiple sclerosis patients and healthy controls: test-retest reproducibility and focal visualization of active white matter areas[END_REF] or [ 18 F]DPA714 [START_REF] Abourbeh | Imaging microglial/macrophage activation in spinal cords of experimental autoimmune encephalomyelitis rats by positron emission tomography using the mitochondrial 18 kDa translocator protein radioligand [(1)(8)F]DPA-714[END_REF] all bind specifically to the TSPO complex, with different levels of affinity. Studies conducted using [ 11 C]PK11195 have found a significant increase in tracer uptake in widespread areas of periventricular and subcortical WM [START_REF] Rissanen | In Vivo Detection of Diffuse Inflammation in Secondary Progressive Multiple Sclerosis Using PET Imaging and the Radioligand (1)(1)C-PK11195[END_REF] suggesting diffuse infiltration by activated microglia at the SPMS stage of the disease. This technique was also able to detect the active inflammatory rim of chronic smoldering plaques. Giannetti et al. demonstrated the clinical significance of widespread microglial activation, showing that this phenomenon was detectable using PET with [ 11 C]PK11195 in the NAWM of patients with CIS and more pronounced in those who later developed CDMS (figure 9) [START_REF] Giannetti | Increased PK11195-PET binding in normal-appearing white matter in clinically isolated syndrome[END_REF].

Grey matter involvement

As studies have been limited in their ability to correlate neurological disability to focal WM pathology, the focus shifted not only to diffuse WM abnormalities but also to the possibility of associated GM damage. GM involvment in MS has perplexed scientists for many decades. Dawson's question "Is then, the process that attacks the cortex different in its nature and origin from that which affects the rest of the central nervous system?" still resonates, though histopathological and neuroimaging studies have provided great insight into the nature, extent and chronology of GM pathology in MS.

Grey matter demyelination

Unlike WM lesions, demyelination of cortical neurons is not visible macroscopically in post-mortem samples. In their seminal study, [START_REF] Brownell | The distribution of plaques in the cerebrum in multiple sclerosis[END_REF] [START_REF] Brownell | The distribution of plaques in the cerebrum in multiple sclerosis[END_REF] showed that about 22% of all brain lesions were located at least partly in the cerbral cortex and an additional 4% in the deep grey matter (DGM) structures. Improvement of immunocytochemical staining of myelin proteins has allowed more reliable investigation of GM demyelination, which has proven even more extensive than initially suspected. Recent pathological studies reported that the extent of GM demyelination often exceeds that of the WM in progressive patients [START_REF] Gilmore | Regional variations in the extent and pattern of grey matter demyelination in multiple sclerosis: a comparison between the cerebral cortex, cerebellar cortex, deep grey matter nuclei and the spinal cord[END_REF], reaching up to 68% in extreme cases [START_REF] Kutzelnigg | Cortical demyelination and diffuse white matter injury in multiple sclerosis[END_REF]. Grey matter demyelination is particularly extensive in the spinal cord, cerebellum, cingulate gyrus [START_REF] Gilmore | Regional variations in the extent and pattern of grey matter demyelination in multiple sclerosis: a comparison between the cerebral cortex, cerebellar cortex, deep grey matter nuclei and the spinal cord[END_REF], thalamus [START_REF] Vercellino | Demyelination, inflammation, and neurodegeneration in multiple sclerosis deep gray matter[END_REF] and hippocampus [START_REF] Dutta | Hippocampal demyelination and memory dysfunction are associated with increased levels of the neuronal microRNA miR-124 and reduced AMPA receptors[END_REF], regions that are of particular relevance to MS symptomatology, both physical and cognitive.

Some have suggested that GM demyelination could, by impacting neuronal gene expression, reduce function in affected areas [START_REF] Dutta | Hippocampal demyelination and memory dysfunction are associated with increased levels of the neuronal microRNA miR-124 and reduced AMPA receptors[END_REF].

Lesions found in the MS grey matter differ strikingly from their WM counterparts.

Lymphocyte infiltration, complement deposition, and BBB disruption, all typical pathological hallmarks of WM lesions, are not usually found in cortical lesions (CLs).

Different types of CLs (figure 10) have been described according to their location and extent and include: leukocortical, intracortical and subpial [83] [84]. Leukocortical lesions consist of WM lesions which extend into the GM. Intracortical lesions project along vessels within the cortical ribbon. Subpial lesions are bandlike plaques which extend from the pial surface into the cortical layer 3 or 4 and can involve several gyri. In DGM structures, lesions are more often mixed GM/WM lesions (about 60%) [START_REF] Vercellino | Demyelination, inflammation, and neurodegeneration in multiple sclerosis deep gray matter[END_REF],

whereas subpial lesions are more frequent at the cortical level. At the earliest stages of the disease, leukocortical lesions were found to be the most frequent and inflammatory in nature [START_REF] Lucchinetti | Inflammatory cortical demyelination in early multiple sclerosis[END_REF], with predominantly perivascular CD3+ and CD8+ T-cell infiltrates and less commonly B-cell infiltrates. These differ from cortical lesions found at the latter stages of the disease, which are more frequently subpial and less inflammatory. Whether inflammation is a prerequisite to GM demyelination still remains unsettled.

It has been hypothesized that GM demyelination, and in particular subpial lesions, could be due to a myelinotoxic factor diffusing from the meninges. Subpial cortical lesions are often topographically related to mononuclear inflammatory infiltrates of the meninges in both early and late-stage MS [58] [86]. The presence of these meningeal B-cell follicles has been associated with more extensive cortical damage and a more severe disease course [START_REF] Magliozzi | Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology[END_REF].

The development of new MRI techniques sensitive to GM demyelination has allowed us to appreciate the clinical relevance of these lesions from the early stages of the disease.

Sequences such as double-inversion recovery (DIR) or phase-sensitive inversion recovery have improved the detection of such lesions, and can be implemented on clinical grade scanners (figure 11). Although MRI techniques have become more sensitive to cortical demyelination, many cortical lesions remain undetected using MRI, especially with sequences available for routine practice. At 1.5T, only 18% of cortical lesions are detected when compared to histopathology [START_REF] Seewann | Postmortem verification of MS cortical lesion detection with 3D DIR[END_REF] Higher field strength or combination of sequences can improve detection [89] [90] [91] [92]. Jonkman and colleagues recently reported that T2-weighted sequences at ultra-high field (7T) allowed prospective detection of 28% of cortical lesions [START_REF] Jonkman | Ultra-High-Field MRI Visualization of Cortical Multiple Sclerosis Lesions with T2 and T2*: A Postmortem MRI and Histopathology Study[END_REF]. Visualization of band-like subpial lesions still remains challenging [START_REF] Yao | 7 Tesla magnetic resonance imaging to detect cortical pathology in multiple sclerosis[END_REF] and availability of such techniques is currently limited. It is thus only the "tip of the iceberg" that can be seen and quantified using new MRI techniques [START_REF] Seewann | Imaging the tip of the iceberg: visualization of cortical lesions in multiple sclerosis[END_REF]. But this "tip of the iceberg" still holds clinical significance. MRI measures of cortical demyelination have shown that GM lesions play an important role in physical and cognitive deficits (table 2). Cerebellar cortical lesion load also correlates with cerebellar function.

The potential contribution of CLs to the diagnostic process was recently suggested by Filippi et al. [96]. The authors found that the presence of at least one CL on baseline DIR MRI improved the identification of patients with CIS at risk of converting to CDMS.

The applicability of new criteria of DIS which would include the presence of CLs remains to be further explored, especially considering the high false positive detection of CLs [88] [93] despite established consensus scoring recommendations [START_REF] Geurts | Consensus recommendations for MS cortical lesion scoring using double inversion recovery MRI[END_REF].

Neurodegeneration

As we have seen previously, degenerative changes in axons within acute WM lesions or NAWM have been well documented. Postmortem studies have also provided evidence of early and evolutive neuronal pathology in the MS grey matter. Peterson and colleagues reported neuritic changes within cortical lesions described as axonal transections, dendritic transections and apoptotic loss of neurons [START_REF] Peterson | Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions[END_REF]. Axonal loss in GM structures is associated with on-going inflammatory activity [83] [81]. Yet, axonal damage in active GM lesions remains much less extensive than in acute active WM lesions. Neuronal death on the other hand was seen in chronic lesions without significant inflammation, suggesting that this phenomenon was not directly linked to the immune insult but might be a consequence of chronic injury. The neurodegenerative changes seen in the MS cortex are more subtle than those described in DGM structures, particularly the thalamus. Cifelli and colleagues reported a 23% reduction in mean neuronal density corresponding to a 35% loss of total neuronal numbers in the MS thalamus compared to controls [110]. Unlike neocortical structures, neuronal density was decreased in both demyelinated and non-demyelinated DGM compared to control DGM, though the loss of neurons may be more pronounced in demyelinated areas [START_REF] Vercellino | Demyelination, inflammation, and neurodegeneration in multiple sclerosis deep gray matter[END_REF]. Neuronal atrophy and morphologic changes were also detected in the MS DGM regardless of myelination status and may precede or accompany neuronal loss. In the hippocampus, neuronal counts were decreased by up to 30% depending on location [111]. Also in the hippocampus, Dutta et Many however argue that WM pathology cannot satisfactorily explain the full extent of diffuse GM damage observed in MS, including pathological changes observed therein [START_REF] Calabrese | Exploring the origins of grey matter damage in multiple sclerosis[END_REF]. Despite a clear degree of relationship, neuronal damage may also occur independently from WM pathology.

GREY MATTER ATROPHY

The cortex represents 65% of brain parenchymal tissue and GM largely drives the atrophic process occuring in MS. GM volume can be measured on conventional MRI sequences with freely available tools among which the most commonly used are FSL (http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/), FreeSurfer (http://freesurfer.net) and SPM (http://www.fil.ion.ucl.ac.uk/spm/). This has tremendously helped the field in understanding the occurrence and chronology of GM tissue loss, as total and regional GM volumes, as well as cortical thickness can be assessed (Figure 17). MS lesions tend to be recognized as GM by the different softwares used in research, a misclassification that may affect GM volume estimation and obscure disease-mediated volume changes. This is why a procedure, called "lesion inpainting" is generally applied before GM segmentation. This procedure replaces lesional voxels with values approximating NAWM intensity [141]. It has been shown to significanly improve accuracy of GM volume and cortical thickness estimation. At the group level, GM atrophy can be seen from the first clinical event as loss of GM There are several limitations however to the use of GM atrophy as a sensitive marker of neuronal damage in MS. First, measures of GM atrophy or cortical thinning do not reflect the entire spectrum of neurodegenerative changes occuring within the MS GM.

Several studies have shown significant alterations in synaptic density or dendritic arborization in animal models of the disease [155], as well as in the MS neocortex [START_REF] Peterson | Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions[END_REF] [105]. These changes are also particularly significant in the hippocampus. Dutta and colleagues reported decreased hippocampal neuronal gene expression, in particular KIF1A, which is involved in anterograde transport of synaptic vesicles to the presynaptic terminal. They found evidence of synaptic pruning even in the absence of significant Another potential issue is that volumetric measurements may be influenced by water content, extracellular matrix, and inflammatory infiltration. Several studies reported robust changes in brain volumes between dehydrated and rehydrated states [159] [160].

A volume change of about 0.36% during the dehydration-rehydration interval is well above previously reported scan-rescan reproducibility errors and close to a recently proposed pathological "cut-off" for volume loss of 0.4% [161]. Furthermore, it can be a complicated task to distinguish true atrophy from "pseudoatrophy", a significant loss of brain volume, which may occur after initiation of anti-inflammatory therapy, and a phenomenon that is not yet fully understood [162]. Progressive and widespread DTI alterations have been demonstrated in the MS GM, though these changes have shown conflicting directionality depending on the study. MD consistently increases across studies, while FA has been found to either decrease or increase. The discrepancy has been explained by possible differences in GM For the reasons cited above, DTI appears to be a sensitive yet non-specific marker of GM damage in MS. This observation along with the difficulties associated with image noise, echo planar imaging (EPI)-induced distortions and partial volume averaging in large voxels, challenge the application of DTI measurements for diagnostic or prognostic purposes at the individual level in MS.

QUANTITATIVE MRI

Magnetization Transfer Imaging (MTI)

MTI is a MRI technique that generates contrast based on the phenomenon of magnetization exchange between the unbound protons in free water and the protons bound to macromolecules. Changes in magnetization transfer ratio (MTR, which reflects the exchange rate of magnetisation transfer between the two proton pools) have been

Proton Magnetic Resonance Spectroscopy

Proton Magnetic Resonance Spectroscopy ( 1 H-MRS) allows quantification of different metabolites in brain tissues (figure 19). It can be used to measure levels of N-acetyl Aspartate (NAA), a metabolite long recognized as an indicator of neuronal and axonal function. Based on immunohistochemical findings NAA is present in most neuronal cell populations, but the intracellular concentration appears to vary greatly between neuronal groups [187]. After synthesis by mitochondria, NAA is exported to the neuronal cytoplasm, where it remains present in high concentration [188]. Its function has yet to be fully established.

NAA levels can be measured using 1 H-MRS either within small volumes (single voxel 1 H-MRS, figure 14) or across larger areas (Chemical Shift Imaging, CSI). Hunter's angle, drawn from the myoinositol peak to NAA peak is a visual way to assess normality of the NAA spectrum. It normally averages 45° from the x-axis. NAA or other metabolite concentrations are usually expressed as a ratio (relative quantification) rather than as absolute concentrations. In relative quantification, one metabolite peak is used as the concentration standard. A classic example is the ratio NAA/ Creatine. One of the limitations of such quantification method is that changes in the ratio can reflect changes in the concentration of the numerator, the denominator, or both and are usually more difficult to interpret than absolute concentrations. NAA levels have been found to be reduced by 10-20% on average in the thalamus of RRMS patients compared to controls [110] [190]. Geurts and colleagues also reported significant decrease in thalamic NAA in PPMS patients [191]. Lower NAA/Cr values were found in the posterior cingulate gyrus of RRMS patients when compared to controls in one study [189]. Though other groups were unable to detect similar metabolic changes in the cortical grey matter [191] [192] [193] [73].

A reduction in NAA levels can occur in case of neuronal loss or damage but can also reflect reversible metabolic dysfunction affecting the mitochondria. Histopathological studies have demonstrated robust correlations between NAA levels and axonal pathology [194]. Although MRS provides unique and specific metabolic information regarding neuronal function and viability, several factors have limited its utility. Among these limitations, the necessary sampling over large voxels, poor spatial resolution, and susceptibility artifacts in regions close to bone may explain why MRS has not been more widely used to assess GM and in particular cortical damage in MS, although this technique has been available for many years.

POSITRON EMISSION TOMOGRAPHY

Principles of PET imaging

A PET scanner is an imaging system that provides analytical tomographic measurements of the tissue concentration of compounds labeled with a positron emitting radionuclide (figure 20). Only minimal or ''tracer doses'' of neuroreceptor radiotracers need to be injected to estimate receptor parameters. After injection, the study subject is placed within the field of view (FOV) of the scanner where several detectors can register incident gamma rays. The radionuclide decays and emits positrons, which then annihilate with an electron to produce two 511 keV gamma rays.

The gamma rays are emitted in opposite directions at approximately 180 degrees from each other and can be detected by the scanner's detectors. The annihilation event is usually very close to the site of positron emission because the emitted positrons rapidly lose their energy in tissue. The distance the positrons travel before annihilation is small, typically less than 2 mm. The detectors are connected with fast timing circuits that look for two simultaneous or "coincident" events on opposite sides of the head. Detection of the two coincident gamma rays defines a line, which intersects the position of the annihilation event. These "coincidence events" can be stored in arrays corresponding to projections through the patient. The raw data collected by the PET scanner is then mathematically reconstructed to produce tomographic images of tissue radioactivity concentration. Coregistration of MRI and PET data enables the interpretation of functional data with localizing high-resolution structural information.

[ 18 F]-fluorodeoxyglucose (FDG) PET and neuronal metabolism

Glucose is the brain's main substrate for energy supply.

PET with [ 11 C]FMZ in neurological disorders

The Partial Saturation Method, a non invasive quantification technique

Despite promising findings, the more widespread application of Therefore there is a crucial need for the development of imaging techniques, aimed at visualizing and quantifying neuronal damage in MS.

GABAA receptor density measured in vivo by PET with [ 11 C]FMZ has been reported as a specific marker of neuronal integrity and has allowed to estimate neuronal damage in several neurological and psychiatric disorders. We hypothesized that this technique could provide insights into the extent of neuronal pathology in MS patients.

The aims of this pilot study are as follow:

-To quantify neuronal damage with [ 11 C]FMZ PET at different stages of the disease using a non-invasive quantification method.

-To investigate the relationship between neuronal injury and macroscopic WM damage.

-To assess the clinical significance of this technique by determining the correlation between [ 11 C]FMZ PET metrics and measures of physical and cognitive performance.

MATERIAL AND METHODS

This study was approved by the local Ethics committee. All subjects gave written consent for participation.

Subjects

A total of 10 healthy volunteers and 20 MS patients were initially included in the study.

Inclusion criteria for MS patients were as follow:

- Women that were childbearing or breastfeeding were excluded. All females were advised to use an efficient contraceptive method and a plasmatic pregnancy test was performed before each PET examination.

One healthy volunteer showed incidental abnormalities on MRI suggestive of small vessel disease, one volunteer and one SPMS patient withdrew their consent before the end of the study and one RRMS subject failed to complete the MRI exam. Therefore the final population analyzed comprised 8 healthy volunteers and 18 subjects with MS ( 9RRMS and 9 SPMS).

Clinical Evaluation

Neurological and neuropsychological assessments were performed at time of MR imaging.

Physical disability was evaluated using:

-

Magnetic Resonance Imaging Data Acquisition

MRI was acquired on the same day as the clinical visit.

MRI was obtained for all participants on the same 1.5T Signa scanner (General Electrics Medical Systems) including:

1) 3D-Fast Spoiled Gradient Recalled Echo (FSPGR) T1-weighted: TE= 2.2, TI=600, TR= 3000 ms, FOV= 240x260 mm, matrix 192x256, slice thickness:1.3 mm 124 slices;

2) 2D-T2 weighted (FSE): TR=2920, TE=103, FOV=240x260 mm, slice thickness=4.0 mm, matrix 256x160;

3) 2D-Fast fluid attenuated inversion recovery (FLAIR) TR=8002, TE=152, FOV=240 mm, slice thickness=4.0 mm, matrix 256x256.

PET acquisition and quantification

Processing

Processing steps are summarized in figure 24.

WM lesion segmentation and inpainting

Using a semi-automated segmentation technique based on local thresholding (Jim version 3, Xinapse Systems, UK, http://www.xinapse.com), MS lesions were delineated by a single experienced observer on the 2D-T2 images using the coregistered information from FLAIR images. WM T2 lesion volume was calculated based on the T2 lesion masks provided by Jim Software, then normalized for headsize of each subject.

Prior to the inpainting process, lesions were also segmented on 3D-FSPGR images in the native space using the coregistered information from T2-weighted and FLAIR images.

We then applied an automated inpainting technique that replaces lesional voxels with values derived from the intensity distribution of the white matter outside of lesions

[141].

Grey matter segmentation and parcellation

Cortical surface reconstruction and volumetric segmentation were performed using the "inpainted" 3D-FSPGR volumes with the Freesurfer v5.1.0 image analysis suite, (http://surfer.nmr.mgh.harvard.edu/). The technical details of these procedures are described in prior publications [223][224][225][226]. The segmentation of GM volumes and of the GM/WM interface for each subject was inspected and reprocessed after manual or automatic adjustments if errors were found.

Registration

The parametric images of [ 11 C]FMZ Bmax were registered to 3D-FSPGR images using a boundary-based registration method [227]. We visually checked all subjects to assess proper registration.

Volume of interest (VOI) analyses

Freesurfer segmentation and cortical parcellation with the Desikan-Killiani atlas label over 50 separate brain structures (Annex C).

Results for total GM (TGM), cortical GM (CGM), lobar GM (frontal, cingulate, parietal, temporal, occipital and insula) and subcortical GM (thalamus proper, caudate, pallidum, putamen, hippocampus and amygdala) are presented here.

In the cortical GM, we calculated the mean thickness of each lobe as well as the mean global cortical thickness.

The volume of each subcortical GM structure was calculated from the 3D-FSPGR images and normalized for head size.

For each volume of interest, we calculated the mean Bmax, or density of BZD receptors, as well as the total amount of [ 11 C]FMZ binding sites defined as the product of the Bmax by the total volume of the structure, then normalized by head size.

Surface based analyses

As the cortical thickness can vary substantially throughout the cortex, PET signal was registered on each individual surface and mapped along the midline of the cortical ribbon. Signal was then smoothed along the surface using a 2D 10 mm full width at half maximum (FWHM) Gaussian kernel. Data were then registered for all subjects to a surface template using FreeSurfer. The relationship between cortical thickness and Bmax across subjects both in the patient group and the control group, and between cortical [ 11 C]FMZ binding sites and WM T2 lesion load were evaluated using Spearman's rank correlation.

Correlation between cortical Bmax and PASAT scores was also computed on a vertexby-vertex basis for all patients using a GLM (n=18).

RESULTS

Characteristics of MS patients and healthy controls

Demographic and clinical characteristics of MS patients and controls are presented in table 3.

A total of 8 healthy controls and 18 MS patients were included in the study. The patients were divided in 2 groups: RRMS (n=9) and SPMS (n=9). The control and MS groups did not significantly differ with regard to age (p=0.85), but RRMS subjects were younger than SPMS subjects. The groups were not matched for sex. We thus took into account both age and sex in our statistical analyses.

The SPMS group had a longer disease duration compared to the RRMS group (24.3 years vs 7.0 years). They also had higher EDSS scores (6.2 vs 2.2). Comparisons of physical and cognitive performance between groups were performed using linear regression analyses adjusting for the effect of age and sex (Table 4). The 25-FW could only be assessed in 14 of the MS patients. 4 patients could not complete the task, as they were wheelchair bound. All subjects performed the rest of the clinical evaluation. We found no difference between patients and controls for the [START_REF] Brownlee | Earlier and more frequent diagnosis of multiple sclerosis using the McDonald criteria[END_REF]

Healthy

[ 11 C]FMZ binding in the grey matter

Reconstructed PET images yielded high resolution whole brain parametric maps of BZD receptor concentration (Bmax) that clearly localized within GM areas. The quality of the output can be assessed on figure 25, which presents parametric maps in a healthy control and in a MS patient.

[ 11 C]FMZ Bmax was 2.5 fold higher in cortical regions compared to subcortical GM: in the healthy control group, median cortical Bmax was 111.7 pmol/ml (88.3-124.8) and median subcortical GM Bmax was 43.5 pmol/ml, (37.4-51.9). suggesting that age did not interfere as a major confounding factor in [ 11 C]FMZ binding estimation (figure 26). However the correlation between age and volumetric measurements was significant both in the cortical and subcortical GM and justified correction for the effect of age in our statistical analyses.

GM atrophy in MS patients

GM atrophy was assessed by means of GM volume estimation in the total GM, cortical GM and in subcortical GM structures. GM volumes were normalized for headsize prior to comparison between groups. The mean cortical thickness was also estimated for the whole cortex and for the different lobes (frontal, parietal, temporal, occipital, insula and cingulate). Cortical thinning was assessed using ROI analyses and surface-based analyses.

When total grey matter and total cortical volumes normalized for headsize were compared between the control group and the MS group, the difference did not reach significance after adjustment for age and sex (figure 27 A,B). The total volume of deep GM structures normalized for headsize was reduced in MS patients compared to HC (figure 27,C), the difference being significant after adjustment for age and sex in the total subcortical GM (p=0.003), the thalami (p=0.0003) and the left caudate nucleus (p=0.02) (figure 27,D).

The mean cortical thickness for the whole cerebral cortex was 2.44 ± 0.08 mm in healthy controls and 2.39 ± 0.11 mm in the whole MS group, with variations depending on cortical location. On ROI analyses, cortical thickness was significantly decreased in MS patients compared to controls in the temporal and occipital lobes bilaterally (figure 27,E). Vertex-by-vertex GLM analyses adjusted for age and sex failed to detect significant cortical thinning.

Reduced [ 11 C]FMZ binding in the MS GM

In cortical structures

Mean [ 11 C]FMZ Bmax was reduced in MS patients compared to controls with a 9.4% decrease in the total GM and a 10% decrease in cortical GM, the difference remaining significant when adjusted for age and sex in the cortical GM (p=0.037, fig 28 A,B).

The cortical parcellation obtained with FreeSurfer allowed a regional analysis of [ 11 C]FMZ Bmax changes, showing for the MS group a significant decrease in the parietal, cingulate, and insular cortices of both hemispheres, and in the left frontal cortex (figure 28,C).The estimated total amount of [ 11 C]FMZ binding sites, defined as the product of the Bmax by the total volume of the structure, then normalized by head size showed a decrease in similar proportion that reached significance in both the total GM (-10.3%, p=0.03) and the cortical GM (-10%, p=0.03) (figure 29 A,B).

Significant negative correlations were found between cortical thickness and cortical

Bmax across cortical regions both in HC (r=-0.86, p= 0.0006) and in MS patients (r=-0.98, p<2.2x10 -16 ) (figure 30,A). But no correlation between cortical thickness and cortical Bmax across subjects was found either in healthy controls (r=0, p=1) or in MS patients (r=0.12, p=0.64) (figure 30,B).

In subcortical structures:

Bmax showed a non-significant trend in the subcortical GM of MS subjects (-8.8%, p=0.32). The difference on ROI analyses only reached statistical significance in the left amygdala (figure 31).

But the total estimated number of [ 11 C]FMZ binding sites per GM structure, showed a significant decrease (figure 32) in the:

-Total subcortical GM (-17%, p=0.008) -Left and right thalami (-23% and -25% respectively, p=0.001) -Left and right hippocampi (-15%, p=0.003 and -19%, p=0.005 respectively)

-Left and right amygdalae (-21%, p=0.001 and -18%, p=0.02 respectively).

Cortical mapping of [ 11 C]FMZ binding changes

It was of interest to see if we could map [ 11 C]FMZ binding changes between groups to determine which cortical regions were more affected by the loss of BZD receptor sites.

This was done using the vertex-by-vertex GLM procedure available in FreeSurfer.

Comparisons were adjusted for the effect of age and sex and corrected for multiple comparisons.

The vertex-by-vertex GLM procedure applied to

Relationship between [ 11 C]FMZ binding and white matter lesions load

To determine the impact of focal WM damage on [ 11 C]FMZ binding, we evaluated the correlations between PET measures and global T2 lesion load (figure 35).

T2 lesion load was estimated for each subject and averaged at 19,550 mm 3 in RRMS patients (2,627-37,965 mm 3 ) and 40,167 mm 3 in SPMS patients (5,865-66,596 mm 3 ).

There was a significant negative correlation between T2 lesion volume fraction and

Relationship between [ 11 C]FMZ binding and clinical metrics

It was of interest to evaluate the clinical relevance of our PET metrics by assessing their relationship with physical and cognitive performance as part of an exploratory analysis.

Relationship between clinical scores and cortical [ Correlations between cortical Bmax and PASAT performances were also tested at the surface-based level to determine the cortical regions associated with PASAT performance (figure 36, table 7). Significant positive correlations were detected in:

-Right posterior cingulate -Right precentral -Left postcentral gyri

No cluster with negative correlation was detected. 37). A trend was found between FA within cingulate white matter tracts and posterior cingulate Bmax (rho=-0.40, p=0.097). Considering the short disease duration, follow-up of these subjects will be very informative to determine the nature and chronology of a relationship between these two phenomena.

Relationship between GM demyelination and neurodegeneration

The in vivo relationship between cortical neuronal damage and cortical demyelination also needs to be investigated. Studies of autopsy material have determined that demyelination is extensive in the GM of patients with MS, especially in the secondary progressive phase of the disease [START_REF] Bo | Intracortical multiple sclerosis lesions are not associated with increased lymphocyte infiltration[END_REF]. The seminal work by Peterson et al. [83] identified axonal transections, dendritic transections and neuronal deaths by apoptosis within areas of cortical demyelination in MS brains. 39). No significant difference was found between NAGM Bmax in MS patients compared to healthy controls. These findings suggest that a significant level of neuronal injury might occur in conjunction with demyelination at the early stages of the disease, and be limited to areas of GM demyelination. Neuronal injury in demyelinated GM may relate to acute damage to axo-dendritic processes secondary to inflammation [START_REF] Vercellino | Demyelination, inflammation, and neurodegeneration in multiple sclerosis deep gray matter[END_REF]. Indeed, Lucchinetti et al.

reported significant inflammation within early cortical lesions, which differed from chronic GM lesions in that respect. The authors also described neuronal damage occurring on a background of inflammation at this stage supporting the notion of a secondary neurodegenerative process, in the context of local inflammation in early MS [START_REF] Lucchinetti | Inflammatory cortical demyelination in early multiple sclerosis[END_REF]. The inflammatory milieu may alter expression and phosphorylation of AMPA receptors or downregulate genes involved in glutamatergic neurotransmission, thus promoting neuronal and synaptic damage via glutamate accumulation and excitotoxicity [263]. As the disease evolves, inflammation becomes much less pronounced within cortical lesions and the relationship between GM demyelination and neuronal/synaptic injury may not be as exclusive. Longitudinal follow-up of our subjects as well as assessment of patients at different stages of the disease may provide greater insights on the evolving interplay between these two processes.

Other aspects

Other mechanisms at play in the MS grey matter have been proposed. Evidence of cerebral hypoperfusion in MS is currently gaining some attention as it may negatively impact an already precarious energy balance in neuronal cells [130]. From a clinical standpoint, reduction in cerebral blood flow (CBF) has been associated with cognitive deficits and fatigue in MS patients [264] [134].

In a recent work led with the team of Dr Wolinsky at the University of Texas at Houston and recently presented at ECTRIMS, I was able to show that hypoperfusion is a widespread phenomenon in the MS GM, which can be assessed using non-invasive MRI techniques. We performed pseudo-continuous arterial spin labeling (pCASL) MRI in 34

MS patients (5 CIS, 24 RRMS, 5 SPMS) and 19 healthy controls and used a region-ofinterest and a surface-based approach to evaluate cortical and subcortical GM blood flow differences between groups. ROI analyses showed diffuse decrease of CBF in MS neocortex and DGM structures compared to controls, in proportions that surpassed that of atrophy measures (figure 40). Thalami and Pallidi showed the most significant decrease in CBF among DGM structures (-29% and -25% respectively, p<0.005 adjusting for age and sex). Fronto-temporal cortices were among the most affected in surface-based analyses (figure 41, table 8). We also found a moderate yet significant positive relationship between cortical hypoperfusion and cortical volume at the crosssectional level (rho=0.36, p=0.03).

Manipulation of 2D pCASL data can be challenging and care must be taken to apply partial volume effect correction algorithms to limit cross-tissue contamination. Surfacebased analyses present a definite advantage in this regard as we have discussed earlier.

The interpretation of our results also requires caution. We will need to establish that cerebral hyperperfusion in MS does not simply reflect contemporaneous loss of neurons and atrophy but is rather an early and integral feature of MS pathophysiology. Debernard et al. [131] provided evidence of cerebral hypoperfusion at the very early In our pilot cohort of MS patients, we had set out to compare [ 11 C]FMZ binding, glucose metabolism evaluated using PET with [ 18 F]FDG and multimodal MRI metrics in the thalamus of MS patients and controls (figure 42). In agreement with prior findings, we found higher FA values in MS patients compared to healthy controls. This increase, even though not significant, was more pronounced in progressive patients. MTR modifications were minor in the thalamus. This is not a surprising finding since MTR has been shown to be mainly affected by myelin content rather than neuronal damage.

Spectra analysis showed a slight decrease in NAA/Cr ratio in progressive patients, due in equal proportions to a decrease in NAA (neuronal injury) and increase in Creatine (gliosis). There was a moderate decrease in neuronal metabolism in RR patients that could reflect early disruption between thalamus nuclei and the cortex as suggested by Derrache et al. (2006). However this difference did not reach statistical significance. No change in metabolism was observed in progressive patients. This might be explained by the significant volume reduction observed in this structure, which could induce an artificial increase in FDG uptake. Please note that no arterial blood sampling for PET quantification was performed. Overall, changes measured with [ 11 C]FMZ were of much greater amplitude than those noted with multimodal MRI or PET using FDG, suggesting that [ 11 C]FMZ PET may provide a more sensitive quantitative marker of thalamic injury.

This approach and correlations between the different metrics need to be validated in a larger group of MS patients.

LIMITS OF PRESENT STUDY

The work presented here has some limitations. The sample of MS patients and controls is small and rather heterogeneous. The sex ratio between patients and controls was not matched. Similarly, while the control and MS groups did not differ with regard to age, the RRMS subjects were significantly younger than their SPMS counterparts.

Consequently, the results of this pilot study will need to be replicated in a larger and homogeneous cohort of MS patients, with adequate matching to a control population.

The study of early MS patients (<5 years) is attractive as GM atrophy at this stage is not prominent and may not confound measurements of the density of BZD receptors using

Résumé en anglais :

Multiple sclerosis (MS) has long been regarded as an inflammatory demyelinating disorder of the white matter. But post-mortem studies have recently shed light on the extensive involvement of the grey matter (GM). Neuronal damage, characterized by synaptic and dendritic loss as well as neuronal apoptosis, is thought to be a major substrate of physical and cognitive deterioration in MS patients. There is a crucial need for new imaging techniques able to specifically assess neuronal damage in MS.

Using positron emission tomography (PET) with [ 11 C]flumazenil ([ 11 C]FMZ), an antagonist of the central benzodiazepine site located within the GABAA receptor, and a non-invasive quantification method, we measured and mapped neurodegenerative changes in the GM of patients with MS at distinct disease stages. A cohort of 18 MS patients was compared to 8 healthy controls and underwent neurological and cognitive evaluations, high-resolution dynamic [ 11 C]FMZ PET imaging and brain MRI. PET data were evaluated using a region of interest and a surface-based approach.

[ 11 C]FMZ binding was significantly decreased in the cortical and subcortical GM of MS patients compared to controls. These changes were significant in both progressive and relapsing-remitting forms of the disease and correlated moderately with white matter lesion load. [ 11 C]FMZ cortical binding was also associated with cognitive performance. This pilot study is the first to quantify in vivo the neurodegenerative changes occurring in MS. Our results show that PET with [ 11 C]FMZ could be a promising and sensitive quantitative marker to assess and map the neuronal substrate of GM pathology in MS.

Key words: multiple sclerosis, neurodegeneration, PET, MRI, Flumazenil, GABAA receptors.

FiguresFIGURE 1 :

 1 Figures

FIGURE 2 :

 2 FIGURE 2: MRI OF THE BRAIN POST-GADOLINIUM INJECTION IN ONE MS PATIENT .................................................

FIGURE 3 :

 3 FIGURE 3: CERVICAL CORD LESION IN MS ON SAGITTAL MRI ...............................................................................................

FIGURE 4 :

 4 FIGURE 4: CLINICAL PHENOTYPES OF MS ........................................................................................................................................

FIGURE 5 :

 5 FIGURE 5: DURATION OF THE 2 PHASES OF MS (FROM LERAY ET AL., BRAIN, 2010) ...............................................

FIGURE 6 :

 6 FIGURE 6: ILLUSTRATION OF MECHANISM OF ACTION FOR MULTIPLE SCLEROSIS THERAPIES. .......................

FIGURE 7 :

 7 FIGURE 7: [ 11 C]PIB IN MS. ......................................................................................................................................................................

FIGURE 8 :

 8 FIGURE 8: PATHOLOGY AT DIFFERENT STAGES OF THE DISEASE ......................................................................................

FIGURE 9 :

 9 FIGURE 9: NORMAL APPEARING WHITE MATTER ON PK11195-PET IMAGES. ..............................................................

FIGURE 10 :

 10 FIGURE 10: DIFFERENT TYPES OF CORTICAL LESIONS IN MS. ..............................................................................................

FIGURE 11 :

 11 FIGURE 11: IMPROVED DETECTION OF INTRA-CORTICAL LESIONS ...................................................................................

FIGURE 12 :

 12 FIGURE 12: NEURONAL LOSS IN TYPE III CEREBRAL CORTICAL LESIONS IN MS .........................................................

FIGURE 13 :

 13 FIGURE 13: SYNAPTIC LOSS IN DEMYELINATED HIPPOCAMPUS .........................................................................................

FIGURE 14 :

 14 FIGURE 14: RETROGRADE AND ANTEROGRADE DEGENERATION ......................................................................................

FIGURE 15 :

 15 FIGURE 15: B-CELL FOLLICLE-LIKE STRUCTURES IN MENINGEAL SPACE OF SPMS PATIENT ..............................

FIGURE 16 :

 16 FIGURE 16: OXIDATIVE STRESS-RELATED MECHANISMS OF TISSUE INJURY IN MULTIPLE SCLEROSIS ..........

FIGURE 17 :

 17 FIGURE 17: EXAMPLE OF FREESURFER FOR GM VOLUME AND CORTICAL THICKNESS ESTIMATION. .............

FIGURE 18 :

 18 FIGURE 18: DIFFUSION TENSOR IMAGING. ......................................................................................................................................

FIGURE 19 :

 19 FIGURE 19: 1 H-MRS ......................................................................................................................................................................................

FIGURE 20 :

 20 FIGURE 20: PRINCIPLES OF PET IMAGING. ......................................................................................................................................

FIGURE 21 :

 21 FIGURE 21: THE PARTIAL SATURATION METHOD ......................................................................................................................

FIGURE 22 :

 22 FIGURE 22: PARAMETRIC IMAGE OF THE [ 11 C]FMZ BMAX FROM A SINGLE EXPERIMENT....................................

FIGURE 23 :

 23 FIGURE 23: STUDY VISITS AND STEPS FROM INCLUSION TO IMAGE PROCESSING. ....................................................

FIGURE 24 :

 24 FIGURE 24: PROCESSING PIPELINE OF PET AND MRI DATA. ..................................................................................................

FIGURE 25 :

 25 FIGURE 25: PARAMETRIC MAPS OF THE BZD RECEPTOR CONCENTRATION .................................................................

FIGURE 26 :

 26 FIGURE 26: CORRELATION BETWEEN [ 11 C]FMZ BMAX AND AGE.........................................................................................

FIGURE 27 :

 27 FIGURE 27: VOLUMETRIC COMPARISONS BETWEEN MS PATIENTS AND CONTROLS ...............................................

FIGURE 28 :

 28 FIGURE 28: ROI ANALYSES OF [ 11 C]FMZ BMAX IN CORTICAL STRUCTURES ...................................................................

FIGURE 29 :

 29 FIGURE 29: ROI ANALYSES OF THE ESTIMATED NUMBER OF [ 11 C]FMZ BINDING SITES. ......................................... FIGURE 30: CORRELATION BETWEEN [ 11 C]-FMZ BMAX AND CORTICAL THICKNESS. ...............................................

FIGURE 31 :

 31 FIGURE 31: ROI ANALYSES OF [ 11 C]FMZ BMAX IN DEEP GM STRUCTURES .....................................................................

FIGURE 32 :

 32 FIGURE 32: ROI ANALYSES OF [ 11 C]FMZ BINDING SITES IN DEEP GM STRUCTURES. .................................................

FIGURE

  FIGURE 34: FREESURFER SURFACE-BASED ANALYSES OF [ 11 C]FMZ BMAX ....................................................................

[ 11 C

 11 ]Flumazenil (FMZ) is an antagonist of the central benzodiazepine (BZD) receptor, a component of the ubiquitous GABAA receptor complex present on neurons throughout the cortical and subcortical grey matter. [ 11 C]FMZ positron emission tomography (PET) could possibly detect and quantify neuronal damage in the grey matter, and provide great insights into the pathophysiology of neurological decline in MS. This has been the working hypothesis of the work presented here. This manuscript will first attempt to present current concepts of WM and GM damage in MS from a histological, immunological and neuroimaging point of view, highlighting the shift from inflammation to neurodegeneration in our understanding of disability progression. We'll underscore the need for new imaging techniques specific of the neuronal compartment to fully capture the neurodegenerative process and its relevance over the course of the disease. In the second part of this work we will propose PET with [ 11 C]-FMZ as potential marker of neuronal damage in MS by presenting the detailed methods and results of a pilot study conducted in relapsing remitting (RRMS) and secondary progressive MS (SPMS). Finally we will discuss our findings in the context of current and sometimes-conflicting ideas and theories of neurodegeneration in MS. We'll aim to expand the scope of this work by presenting future avenues of research relevant to our topic.

Figure 1 :

 1 Figure 1: MS lesions visible as hyperintensities on FLAIR images. Left: Infratentorial lesion found in the pons (arrow). Middle: numerous periventricular lesions, some of which have classic ovoid shape (arrow). Right: subcortical lesions in frontal areas (arrows).

Figure 2 :

 2 Figure 2: MRI of the brain post-gadolinium injection in one MS patient. Periventricular lesions are seen as hyperintense foci on Flair image (left). Recent area of inflammation enhances after gadolinium injection on T1-weighted scan (right)

Figure 3 :

 3 Figure 3: Active cervical cord lesion in MS on sagittal MRI. Lesion is visible as T2 hyperintensity (left) and enhances after Gadolinium injection on T1-weighted scan (right). MS lesions typically extend over <2 vertebral segments.

  of DIT can be achieved on a single MRI with presence of enhancing and non-enhancing lesions, or otherwise by detection of additional lesions on follow-up scans. The revised McDonald criteria have the clear objective of allowing early diagnosis of MS in patients presenting with typical symptoms of CNS demyelination. Still, the risk of over-diagnosing MS remains real and as the Magnetic Resonance Imaging in MS (MAGNIMS) committee recently recommended: "MRI scans should be interpreted by experienced readers who are aware of the patient's clinical and laboratory information" [17].

Figure 4 :

 4 Figure 4: Clinical phenotypes of MS: Relapsing Remitting MS (RRMS, top) without (left) and with (right) accumulation of neurological deficits, Secondary progressive MS (SPMS, middle) without (left) or with (right) clinical exacerbations and primary progressive MS (PPMS, bottom) with linear course (left) or periods of relative stability (right).

Figure 5 :

 5 Figure 5: Duration of the 2 phases of MS (from Leray et al., Brain, 2010). Disability progression during Phase 2 (mean time from EDSS 3 to EDSS 6) in five subgroups defined according to the duration of Phase 1 (mean time from MS clinical onset to EDSS 3) in 718 MS patients who have reached both EDSS 3 and EDSS 6.

Figure 6 :

 6 Figure 6: Illustration of mechanisms of action of currently available or in-trial MS therapies. APC = antigen presenting cell, BBB = blood-brain barrier, C5b-9 = complement complex 5b-9, CNS = central nervous system, FasL = Fas ligand, GA = glatiramer acetate, IFN = interferon, MHC I = major histocompatibility complex I, NO = nitrous oxide, Nrf2 = nuclear factor (erythrocyte derived) related factor 2, S-1-P1 = sphingosine-1-phosphate 1, TCR = T cell receptor, VCAM-1 = vascular cell adhesion molecule 1, VLA-4 = very late antigen 4. From Broadley et al., Journal of Clinical Neuroscience, 2014 [42].

Figure 7 :

 7 Figure 7: [ 11 C]PIB in MS. The uptake of [ 11 C]PIB is lower in MS lesions compared with normal-appearing white matter in patients with MS, suggesting the presence of demyelination. (A) T1-weighted image and (B) corresponding map of [ 11 C]PIB PET binding in a patient with MS, showing lower [ 11 C]PIB uptake in the GM in general compared with WM and lower uptake in a MS lesion (arrow head) compared to normal-appearing WM. (C) Coregistration of images shown in (A) and (B). (D) Time-activity curve of the normalappearing WM (red) and the lesion seen in (A) (blue). PIB=thioflavine-T derivative 2-(40methylaminophenyl)-6-hydroxybenzothiazole.

Figure 8 :

 8 Figure 8: Pathology at different stages of the disease, acute MS (AMS), RRMS, PPMS and SPMS. White matter plaques (green) dominate the disease process in AMS and RRMS, while diffuse WM inflammatory infiltration (blue dots) and GM pathology (orange) are prevalent in PPMS and SPMS. From Kutzelnigg et al., Brain, 2005.

Figure 9 :

 9 Figure 9: Normal appearing white matter on PK11195-PET images co-registered with MRI in three study subjects. The first subject is a healthy control (A) with PK11195 binding potential (BPND) in NAWM of 0.028; the second is a CIS subject without T2 MRI lesions (B), an EDSS score of 3.5 and PK11195 BPND in NAWM of 0.037; the third is a CIS subject with T2 MRI lesions (C), EDSS = 2.0 and PK11195 BPND in NAWM of 0.136. The color scale bar represents the BPND of PK11195. From Giannetti et al., Brain, 2015 [78].

Figure 10 :

 10 Figure 10: Different types of cortical lesions in MS: leukocortical (a), intracortical (b) and subpial (c). (d) highlights the extensive nature of subpial demyelinating lesions which can extend over several gyri. From Calabrese et al., Nature Reviews Neuroscience, 2015 [85].

Figure 11 :

 11 Figure 11: Improved detection of intra-cortical lesions (white arrow) using phase sensitive inversion recovery (PSIR, left) in combination with double inversion recovery (DIR, middle), compared to conventional fluid attenuated inversion recovery (FLAIR, right). Images kindly shared by Dr Flavia Nelson, Associate Professor of Neurology at the University of Texas, Medical School at Houston.

Figure 12 :

 12 Figure 12: Neuronal loss in Type III cerebral cortical lesions in MS. Control (Ctrl) (A) and MS (B) sections immunostained for NeuN demonstrating overall neuronal loss in MS cortex. (C) Antimyelin PLP immunostained section from a patient with MS (B) showing subpial demyelination (original magnification: 100x). High-magnification pictures from cortical layer II (D, E), with matching pictures demonstrating the quality of segmentation scripts (F, G) (digital zoom from original magnification: 100x). From Klaver et al., J Neuropathol Exp Neurol, 2015 [108].

Figure 13 :

 13 Figure 13: Synaptic loss in demyelinated hippocampus. Confocal images of multiple sclerosis (MS) hippocampus immunostained with antibodies specific to MAP2 (red) and synaptophysin (green) show punctuate presynaptic terminals (green) surrounding neurons in CA1 (A, B) and dentate gyrus (C, D) regions in MS myelinated (A, C) and MS demyelinated (B, D) hippocampus. Compared to myelinated MS hippocampi, pre-synaptic terminals were significantly decreased (G) in demyelinated MS hippocampi. From Dutta et al., Annals of Neurology, 2011 [109].

Figure 14 :

 14 Figure 14: Undisturbed neuronal connectivity (upper panel) can be impaired by retrograde degeneration (middle panel) propagating backwards in cortical neurons whose axons have been damages by WM lesions or along WM tracts. The WM tract damage can lead to microglial inflammation (middle panel) and retrograde neuronal cell death (lower panel), as well as anterograde Wallerian degeneration below the damage. From Calabrese, Nature Reviews Neuroscience, 2015 [85].

  a specific role exerted by both meningeal T cells and activated microglia in diffuse axonal loss in the spinal cord[119]. These findings strengthen the hypothesis that meningeal inflammation is implicated in neurodegeneration in MS and contributes to clinical severity and progression.

Figure 15 :

 15 Figure 15: B-cell follicle-like structures in meningeal space of SPMS patient. (A, B) follicle of modest size. (C) Extensive infiltrate filling entire sulcus. From Howell et al. Brain, 2011 [118].

Figure 16 :

 16 Figure 16: Oxidative stress-related mechanisms of tissue injury in multiple sclerosis. (a) Microglia is activated by unknown trigger pathology, the breakdown of the blood-brain barrier, and local and systemic inflammatory stimuli. Microglia releases nitric monoxide and superoxide molecules into the extracellular space. Additional amplification mechanisms involve microglia preactivation via axonal degeneration (d). (b) Iron is physiologically stored within the myelin sheets and liberated into the extracellular space upon demyelination. Extracellular iron amplifies oxidative stress inducing the production of highly reactive hydroxyl radicals. Iron is absorbed by microglia, which show histological signs of cell death under the high iron load and thus may release iron and initiate a second wave of oxidative stress. (c) Mitochondrial DNA (mtDNA) is vulnerable to free radicalmediated damage resulting in mtDNA deletions, which are found in neurons and axons of patients with multiple sclerosis. Mitochondria carrying such mutations are amplified by the clonal expansion in neurons. The mitochondrial respiratory chain is inhibited by covalent modifications caused by free radicals both competitively and irreversibly. A combination of these factors leads to energy failure via decreased ATP production. Exogenous factors, such as free radicals and cyanate delivered by smoking, inhibit mitochondrial function in experimental conditions. From Haider, Oxidative Medicine and Cellular Longevity, 2015 [128]

Figure 17 :

 17 Figure 17: Example of FreeSurfer for GM volume and cortical thickness estimation. After a first step of WM lesion segmentation, a procedure of lesion inpainting is used to limit misclassification of WM lesions as GM. Then the volumetric pipeline can be used for estimation of global or regional GM volumes and region-of-interest (ROI) analyses. The surface reconstruction pipeline allows estimation of cortical thickness.

  neuronal loss and postulated that such changes could underlie the cognitive disturbances experienced by MS patients [109] [156]. Michailidou et al. recently confirmed the extent of synaptic pathology of the MS hippocampus [157], while Novkovic et al. related memory deficits to impairment of synaptic plasticity in experimental autoimmune encephalomyelitis (EAE), a common animal model of MS [158]. These findings clearly support the clinical significance of subtle changes in synaptic density or dendritic arborization occurring in the GM. The changes remain nonetheless undetected by GM atrophy measures [107].

2. 1 .

 1 Diffusion Tensor ImagingDiffusion denotes the random motion of molecules, also called Brownian motion. Water is the most convenient molecular species to study with diffusion MRI, though other metabolites may also be studied. Water molecules move in the brain and interact with many tissue components such as cellular or subcellular membranes or organelles. The distribution of motion of water molecules thus provides clues to the structure and organization of brain tissue. Molecular motion may not be the same in all directions, a phenomenon called anisotropy. Diffusion-weighted MRI only characterizes a single parameter, the diffusion coefficient D. Diffusion tensor imaging (DTI) is used to map and characterize the three-dimensional diffusion of water as a function of spatial location (figure18). The diffusion tensor describes the magnitude, the degree of anisotropy, and the orientation of diffusion anisotropy. Consequently, the diffusion tensor is thought to be a sensitive marker for characterizing both normal and abnormal tissue microstructure[163]. It has been proposed recently as a way to assess GM integrity. The interpretation of diffusion tensor is complex. Common DTI metrics used in MS are fractional anisotropy (FA), which reflect the prevalence of diffusivity along one direction, or Mean Diffusivity (MD) a measure of global water diffusion.

Figure 18 :

 18 Figure 18: Diffusion Tensor Imaging. (A) Representation of an elliptical tensor with 3 main axes. The longest axis is the primary eigenvector (λ1) and reflects the diffusion parallel to fibers ("axial" diffusivity). The other two axes are the second (λ2) and third (λ3) eigenvectors, whose average provides a measure of "radial" or perpendicular diffusivity. (B) DTI color map allows visualization of direction information about diffusion by colorcoding direction information from the eigenvectors.

Figure 19 : 1 H

 191 Figure 19: 1 H-MRS. Left: representative proton MRS spectrum of normal human brain with major peaks of interest depicted. Hunter's angle is represented in red. From Moffett et al. Progress in Neurobiology, 2007 [187]. Right: single voxel acquisition on posterior cingulate gyrus. From Pokryszko-Dragan et al., Journal of the Neurological Sciences, 2014 [189].

Figure 20 :

 20 Figure 20: Principles of PET imaging. From Dr Mirwais Wardak, Brain Imaging with Positron Emission Tomography: Quantification and Biomedical Applications in Alzheimer's Disease and Brain Tumors, UCLA, 2013.

  GABA A receptor density measured in vivo by PET with[ 11 C]FMZ has been reported as a specific marker of neuronal integrity and has allowed, over the past 15 years, to estimate neuronal damage in several neurological and psychiatric disorders. Heiss et al. demonstrated that PET with [ 11 C]FMZ combined with measures of cerebral blood flow could distinguish between irreversibly damaged and viable penumbra tissue [208] [209]. Pascual et al. reported decreased [ 11 C]FMZ binding in the hippocampi and posterior cingulate gyri of patients with Alzheimer's disease [210]. [ 11 C]FMZ binding was decreased precisely in the regions showing the greatest degree of neuronal loss in post-mortem studies of early Alzheimer's disease. In addition, [ 11 C]FMZ hippocampal binding correlated with memory performance. In major depression, decreased binding was found in the limbic parahippocampal and right temporal cortical areas. In the temporal area, [ 11 C]FMZ binding showed a strong inverse correlation with hypothalamic-pituitary-adrenal axis activity [211]. A study using a related tracer [ 123 I]iomazenil in Huntington's disease found a 31% decrease in volume of distribution in the striatum of patients compared to age-matched controls [212]. This change was attributed to loss of binding sites and consequently synapses in these areas. No significant changes in cortical binding were found in these mildly to moderately affected patients. The authors also found strong correlations between [ 123 I]iomazenil binding and clinical functional scores. In epilepsy, PET with [ 11 C]FMZ can be acquired during the interictal period. Several studies have evaluated the clinical utility of [ 11 C]FMZ PET in patients with MRI-negative focal epilepsy. [ 11 C]FMZ PET helped identify surgically relevant abnormalities in about 50% of subjects [207]. The added value of FMZ PET compared to FDG in the context of epilepsy remains however unproven [213].

  [ 11 C]FMZ PET in the research or clinical setting has been limited by highly invasive modeling methods. Standard modeling protocols require serial arterial blood sampling for plasma input function to fit compartmental models. Arterial lines can cause complications such as hematoma, bleeding, and thrombosis and require local anesthetic administration. Other modeling techniques have been proposed to analyze PET data without arterial input function. The simplified reference tissue model [214] model is applicable if a "true" reference tissue exists in which no specific binding of the radioligand occurs. It allows only measurements of the binding potential (BP) which is a combined measure of density of available neuroreceptors (Bmax) and affinity of the ligand to that receptor (Kd). Though proper quantification of changes in receptor density (Bmax) might be important to understand the pathophysiology of neurological disorders. Some investigators have proposed using Scatchard analysis, a common in vitro method based on the equilibrium relation: B/F= (Bmax-B)/KdVR With: B= bound ligand concentration F= free ligand concentration Bmax= BZD receptor density Kd= receptor-ligand affinity VR= reaction volume The plot of B/F vs B gives a straight line whose slope is equal to the inverse of the apparent equilibrium dissociation constant (KdVR) and whose intercept with the B axis gives the receptor density Bmax (figure 21, red line). Using Scatchard analyses eliminates the need for arterial blood sampling but requires separate estimation of bound and free ligand concentrations, as PET data will provide only a sum of these two components. Delforge et al. showed that bound ligand concentration decreases significantly over the usual duration of an experiment after FMZ injection with an average dose between tracer dose and saturation dose [215]. The partial saturation protocol [216] is based on this observation and allows estimation of BZD receptor density and receptor-ligand affinity from a single-injection experiment, without arterial blood sampling.The protocol consists of a coinjection of a[ 11 C]FMZ tracer dose (about 20 mCi) and of a unlabeled FMZ partial-saturation dose (between a tracer dose and a saturation dose)[216]. The choice of the unlabeled FMZ amount is important because it must be sufficiently large to occupy a significant percentage of receptor sites (at least 50% but preferably 70%) but not too large to observe a significant decrease of this percentage during the limited duration of the experiment (1 h at most). Data are analyzed using the usual Scatchard plot (figure21-blue line). The free-ligand concentration F, assumed to be identical in all regions, is estimated from the pons. The pons was initially thought to devoid of BZD receptor sites but was later found to have small but not negligible BZD receptor concentrations (~4.7 pmol/mL)[216]. The percentage of PET-measured ligand concentration in the pons, which corresponds to the ligand bound to the pons receptor sites, can however be estimated as a function of time and of the injected FMZ dose. This curve may then be used in experiments to estimate the free ligand concentration F from the PET-measured pons concentration. The bound ligand concentration B in any region is then obtained by subtracting the free ligand concentration F from the PET-measured concentration. Assuming constant FMZ affinity, parametric images of the Bmax can be obtained[217], which map BZD concentration at each voxel (figure22).

Figure 21 :Figure 22

 2122 Figure 21: Scatchard curve (red) and estimation from PET examination using the partial saturation method (blue)

  Patients had a diagnosis of MS according to McDonald et al. [14] -Either a relapsing-remitting course and EDSS < 4.0 (n=10) or a secondary progressive course and EDSS ≥ 4.0 (n=10) -Able to understand the objectives of the study -Able to give written consent All subjects were free from steroids, cyclophosphamide or mitoxantrone treatment for at least 1 month, and from benzodiazepines or medications known to interact with the BZD receptor for at least 15 days before inclusion in the study. Inclusion criteria for healthy volunteers were: -No history of neurological illness -Able to understand the objectives of the study -Able to give written consent -No abnormalities on brain MRI suggestive of on-going neurological disorder

  The Expanded Disability Status Scale (EDSS, Annex A) -The 25ft timed walk (25-FW) and 9-hole peg test (9-HPT), as part of the Multiple Sclerosis Functional Composite score (MSFC). Each subject also underwent short neuropsychological evaluation, which included: -Paced Auditory Serial Addition Test 3 seconds (PASAT-3), also part of the MSFC, to assess attention, working memory and speed of information processing, -Mini Mental State Examination (MMSE) a general scale sensitive to cortical degeneration -Rey-Osterrieth Complex Figure Test to evaluate visuospatial processing [218] [219]-Stroop Test to evaluate executive functioning[220]

  PET imaging was acquired within 15 days of clinical and MRI examination. All subjects underwent PET with [ 11 C]FMZ on a High Resolution Research Positron Tomograph (HRRT, Siemens Medical Solution, spatial resolution= 2.5 mm), providing 110 slices of 1.1 mm thickness covering the whole brain (Annex B). Ready-to-inject, >99% radiochemically pure [ 11 C]FMZ (ethyl 8-fluoro-5-[ 11 C]methyl-6-oxo-5,6-dihydro-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate or [ 11 C]Ro151788) was prepared from cyclotron-produced [ 11 C]carbon dioxide (Cyclone-18/9 cyclotron, IBA) using a commercially available TRACERLab TM FX-C synthesizer (General Electrics Medical Systems), and based on a process which was formerly performed on a homemade modular system [221]. Participants were positioned in the tomograph with their head maintained using an individually molded headholder. A 6 min brain transmission scan was performed before injection of the radioligand with a 137-Cs point source to correct the emission scan for tissue attenuations. Protocol consisted of a co-injection of carbon-11-labeled FMZ (285 ± 59 MBq) and unlabeled FMZ (10 µg/kg, Anexate ®) in order to partially saturate the BZD receptors. Twelve 3D frames of the entire brain of 5 min each were reconstructed with an accelerated list-mode ordinary Poisson ordered-subset expectation maximization (OP-OSEM) algorithm to lower statistical noise at the voxel level without degrading spatial resolution [222]. The partial saturation protocol [215-217] was used to estimate the BZD receptor concentration (Bmax) for each voxel and voxel-based parametric images of the [ 11 C]FMZ Bmax were computed using an in-house software.

Figure 23 :

 23 Figure 23: Study visits and steps from inclusion to image processing.

Figure 24 :

 24 Figure 24: Processing pipeline of PET and MRI data.

Figure 25 :

 25 Figure 25: Parametric maps of the BZD receptor concentration (Bmax) in a healthy control (A) and MS patient (B)

Figure 26 :

 26 Figure 26: Spearman rank correlation between [ 11 C]FMZ Bmax and age across subjects. No correlation was found between age and [ 11 C]FMZ Bmax in either healthy controls or MS patients.

Figure 27 :

 27 Figure 27: A-C: Box and whisker diagrams of the volume fractions of the total GM (A), cortex (B) and deep GM (C) in healthy controls (HC) and MS patients. D: Volume fraction of subcortical GM structures in HC and MS patients. E: Cortical thickness in the total cortex and cortical lobes (mm) in HC and MS patients. Statistical comparisons were performed using linear regression adjusting for age and sex with a significance threshold of p<0.05.

Figure 28 :

 28 Figure 28: ROI analyses of [ 11 C]FMZ Bmax in cortical structures. A-B: Box and whisker diagrams of [ 11 C]FMZ Bmax in healthy controls (HC) and MS patients in the total GM (A) and cortex (B). C: [ 11 C]FMZ Bmax in HC and MS patients in cortical lobes. Statistical comparison was performed using linear regression adjusting for age and sex with a significance threshold of p<0.05.

Figure 29 :

 29 Figure 29: ROI analyses of the estimated number of [ 11 C]FMZ binding sites. Total FMZ binding sites in the total GM (A), cortex (B) of healthy controls (HC) and total MS patients.

Figure 30 :

 30 Figure 30: Spearman rank correlation between [ 11 C]-FMZ Bmax and cortical thickness across cortical regions (A) and across subjects (B). A: significant negative correlation between mean cortical thickness and [ 11 C]-FMZ Bmax across cortical regions in healthy controls (square marker, dashed regression line) and MS patients (diamond markers, solid regression line). B: no correlation between global cortical thickness and [ 11 C]-FMZ Bmax across subjects in either healthy controls or MS patients.

Figure 31 :

 31 Figure 31: ROI analyses of [ 11 C]FMZ Bmax in deep GM structures A: Box and whisker diagrams of [ 11 C]FMZ Bmax in healthy controls (HC) and MS patients in the deep GM. B: [ 11 C]FMZ Bmax in HC and MS patients in individual deep GM structures. Statistical comparison was performed using linear regression adjusting for age and sex with a significance threshold of p<0.05.

Figure 32 :

 32 Figure 32: ROI analyses of [ 11 C]FMZ binding sites in deep GM structures. A: Box and whisker diagrams of [ 11 C]FMZ total binding sites in healthy controls (HC) and MS patients in the deep GM. B: [ 11 C]FMZ binding sites in HC and MS patients in individual deep GM structures. Statistical comparison was performed using linear regression adjusting for age and sex with a significance threshold of p<0.05.

Figure 33 : 11 C

 3311 Figure 33: Subgroup comparisons Box and whisker diagrams of 11 C-FMZ Bmax (A, B, C) and Total FMZ Binding Sites (D, E, F) in the total GM, cortex, and DGM of healthy controls (HC), RRMS, and SPMS patients.

  [ 11 C]FMZ PET data at the group level detected five clusters of significantly decreased [ 11 C]FMZ Bmax in MS patients compared to controls (figure 34, table 5), localized in: -Left superior parietal gyrus (cluster-wise p-value=CWP= 0.0014) -Right inferior parietal gyrus (CWP= 0.0002) -Right supramarginal gyrus (CWP=0.0028) -Right rostral middle frontal gyrus (CWP=0.0312) -Right pars triangularis (CWP=0.0218). Importantly, no cluster of increased [ 11 C]FMZ Bmax was detected.

Figure 34 :

 34 Figure 34: FreeSurfer surface-based analyses of [ 11 C]FMZ Bmax. A-H: Regions where [ 11 C]FMZ Bmax was decreased in subjects with MS compared to healthy controls, after correction for age and sex. Left hemisphere, top (A-D) and right hemisphere, bottom (E-H). No cluster of increased [ 11 C]FMZ Bmax was found.

[ 11 C

 11 ]FMZ binding sites in:-Cortical GM (r=-0.51, p=0.03) -Subcortical GM (r=-0.67, p=0.003) -Trend only in the total GM (r=-0.46, p=0.06).

Figure 35 :

 35 Figure 35: correlation between T2 lesion load and [ 11 C]FMZ binding sites in the cortical GM, subcortical GM and total GM of MS patients.

Figure 36 :

 36 Figure 36: FreeSurfer surface-based correlations between [ 11 C]FMZ Bmax and PASAT performance. Visual representation of regions where [ 11 C]FMZ Bmax correlates positively with PASAT scores. Left hemisphere, top (A-B) and right hemisphere, bottom (C-D).

Figure 37 :

 37 Figure 37: Correlations between T2 lesion load and NAWM FA within 3 white matter tracts (thalamo-prefrontal, cingulate and cortico-spinal) and [ 11 C]FMZ Bmax in anatomically related cortical areas (rostral middle frontal, posterior cingulate, precentral) in early RRMS patients (unpublished data).

Figure 38 :

 38 Figure 38: Processing steps undertaken to analyze and compare Bmax within DIR-visible cortical lesions (CLs) and normal appearing cortical GM (NAGM).

Figure 39 :

 39 Figure 39: Comparison of mean [11C]FMZ Bmax in cortical NAGM (cNAGM) and within DIR-visible cortical lesions (-16.4%, p=0.023).

Figure 40 :

 40 Figure 40: ROI analyses of cerebral blood flow (CBF, mL/100g/min) in deep GM structures (top) and cortical lobes (bottom). In DGM structures, maximum decrease of CBF was found in the Thalami (-29%, p=0.00005 and -28%, p=0.0002) and Pallidi (-25%, p=0.003 and -24%, p=0.003). Global cortical CBF was 44.0 mL/100g/min (±10.6) in MS patients vs 50.4 mL/100g/min (±9.8) in controls (-13%, p=0.01). Most cortical structures were hypoperfused in MS patients.

Figure 41 :

 41 Figure 41: Surface-based comparisons CBF between MS patients and healthy controls. The vertex-by-vertex GLM procedure applied to CBF data at the group level (after adjustment for the effect of age and sex and correction for multiple comparisons) detected eight clusters of significantly decreased blood flow in MS patients compared to controls. No cluster of increased CBF was detected.

4 .

 4 FUTURE ROLE OF [ 11 C]FMZ PET IN MSWith this pilot work, we have shown that PET with [ 11 C]FMZ could be performed and adequately quantified in a non-invasive manner in MS patients to detect early neuronal injury to the GM. PET has also been used to investigate underlying pathophysiology of neuroinflammation, demyelination and remyelination in MS. In the particular case of [ 11 C]-labeled tracers, the need and high cost of on-site cyclotron production of isotopes has limited the availability and widespread application of PET. Fluorinated analogs [269] have been proposed and should enable the integration of FMZ PET in clinical practice or for evaluation of novel therapies targeting the neuronal compartment. Meanwhile, [ 11 C]FMZ PET has the potential to inform our understanding of nonconventional MRI techniques in the assessment of neuronal pathology. As a specific marker of the neuronal compartment, [ 11 C]FMZ may be used to evaluate the validity and specificity of neuroimaging techniques in vivo. It may also be used to test mechanistic hypotheses by combining imaging approaches, in which the strengths of MRI may add to those of PET.

Figure 42 :

 42 Figure 42: Patients vs controls comparisons of [ 11 C]FMZ, [ 18 F]FDG and multimodal MRI metrics in the thalamus. Unpublished data from our pilot cohort.

[ 11 C

 11 ]FMZ PET. This will also help us elucidate the early phenomena at play in the MS GM.The cross-sectional design of this work can help us appreciate the correlations between neuronal damage and clinical metrics at one point in time. In order to fully understand the temporal relationship between early neuronal damage and disability in MS, longitudinal follow up of MS patients will be necessary. This is also true to understand the interplay between neuronal damage and other aspect of MS pathophysiology such as WM focal lesions, WM diffuse pathology or GM demyelination. With these objectives in mind, we have continued to enroll in new longitudinal studies of early RRMS and PPMS patients aiming to assess the prognostic value of [ 11 C]-FMZ PET and the relationship between neuronal damage and other important processes as the disease evolves.Finally, some technical limitations need to be brought to attention. While we have taken steps to limit the influence of partial volume effect in the study of cortical [ 11 C]-FMZ binding, by means of surface-based smoothing and sampling of PET measures halfway between the white and the pial surface in our surface based analyses, new processing techniques should allow us to further limit its unavoidable impact on PET data. Steps will be taken to investigate this issue. Overall this work highlights the potential of [ 11 C]FMZ PET as a new biomarker of neuronal pathology in MS, which might lead to greater insights into the neurodegenerative mechanisms involved in MS. Further longitudinal studies will determine the chronology of [ 11 C]FMZ binding in MS, the prognostic value of this technique and the interplay between neuronal damage and other aspects of MS pathophysiology. [ 11 C]FMZ PET-derived metrics may also be of great interest for trials evaluating neuroprotective drugs. The high cost of PET examination together with the restricted availability of [ 11 C]FMZ could impede a large application of this technology in a very near future. The production of a fluorinated analog of this tracer should facilitate such clinical applications, and allow innovative pathophysiological studies. Combining [ 11 C]FMZ PET with advanced MRI sequences will help clarify the relative contribution of white matter or cortical demyelination to neuronal damage and help evaluate the impact of neuronal damage on structural and functional brain connectivity. FMZ PET studies may improve our understanding of the neuronal substrate of cognitive deficits in MS. By investigating predominantly inhibitory neurotransmission through GABAA receptors, [ 11 C]FMZ PET could provide insights on functional mechanisms underlying MS-related cognitive dysfunction: cortical neuronal synchrony, which is regulated by GABAergic neurotransmission, is considered mandatory for cognitive processing [277], and could emerge as a key substrate for the pathophysiology of MS cognitive deficits, as previously suggested in schizophrenia or Alzheimer's disease [278] Imaging the neurodegenerative component of a demyelinating disorder : multiple sclerosis.

TABLE 1 :

 1 34: FREESURFER SURFACE-BASED ANALYSES OF [ 11 C]FMZ BMAX ....................................................................FIGURE 35: CORRELATION BETWEEN T2 LESION LOAD AND [ 11 C]FMZ BINDING SITES .......................................... FIGURE 36: FREESURFER SURFACE-BASED CORRELATIONS BETWEEN [ 11 C]FMZ BMAX AND PASAT PERFORMANCE. .................................................................................................................................................................................FIGURE 37: CORRELATIONS BETWEEN T2 LESION LOAD AND NAWM FA WITHIN 3 WHITE MATTER TRACTS ................................................................................................................................................................................................................FIGURE 38: PROCESSING STEPS UNDERTAKEN TO ANALYZE AND COMPARE BMAX WITHIN DIR-VISIBLE CORTICAL LESIONS AND CORTICAL NAGM .......................................................................................................................FIGURE 39: COMPARISON OF MEAN [11C]FMZ BMAX IN CORTICAL NAGM AND WITHIN DIR-VISIBLE CORTICAL LESIONS .......................................................................................................................................................................FIGURE 40: ROI ANALYSES OF CEREBRAL BLOOD FLOW ...................................................................................................... 2010 REVISED MCDONALD CRITERIA FOR DIAGNOSIS OF MS (FROM POLMAN ET AL., ANNALS OF NEUROLOGY, 2010) .........................................................................................................................................................................

FIGURE 41: SURFACE-BASED COMPARISONS CBF BETWEEN MS PATIENTS AND HEALTHY CONTROLS. .....

FIGURE 42: PATIENTS VS CONTROLS COMPARISONS OF [ 11 C]FMZ, [ 18 F]FDG AND MULTIMODAL MRI METRICS IN THE THALAMUS. ..................................................................................................................................................

Tables

TABLE 2 :

 2 CLINICAL RELEVANCE OF CORTICAL LESIONS USING MRI. ................................................................................

TABLE 3 :

 3 DEMOGRAPHIC AND CLINICAL CHARACTERISTICS OF HEALTHY CONTROLS AND MS PATIENTS

TABLE 4 :

 4 CLINICAL MEASURES OF PHYSICAL AND COGNITIVE PERFORMANCE FOR HEALTHY CONTROLS AND MS PATIENTS. ..........................................................................................................................................................................

TABLE 5 :

 5 CLUSTERS OF DECREASED CORTICAL [ 11 C]FMZ BMAX IN MS PATIENTS COMPARED TO HC. ...........

TABLE 6 :

 6

CLINICAL CORRELATIONS ................................................................................................................................................... TABLE 7: GLM CLUSTERS OF REDUCED CORTICAL [ 11 C]FMZ BMAX IN PATIENTS WITH MULTIPLE SCLEROSIS ............................................................................................................................................................................................

TABLE 8 :

 8 GLM CLUSTERS OF DECREASED CBF IN MS PATIENTS COMPARED TO CONTROLS. ............................

Table 1 : 2010 revised McDonald criteria for diagnosis of MS (from Polman et al., Annals of Neurology, 2011) [13]

 1

1.3. Evolution and prognosis 1.3.1. Clinical phenotypes

	Clarity and consistency in defining clinical phenotypes is essential for demographic
	studies, clinical trials and management of therapy in clinical practice. Newly revised
	classification proposed by Lublin and colleagues [21] recommends that patient
	phenotype be assessed on clinical grounds with input from imaging studies when
	needed. According to the new consensus, 3 disease phenotypes can be defined: clinically
	isolated syndrome, relapsing-remitting disease, and progressive disease including
	primary progressive and secondary progressive.

Clinically Isolated Syndromes (CIS) refer to the initial clinical presentation of the disease in patients with symptoms typical of demyelination of the CNS WM tracts, but who fail to show evidence of dissemination in time of the disease process. Patients with CIS may "convert" to definite MS if they meet criteria for DIS and DIT on clinical or radiological grounds. A majority of patients diagnosed with definite MS will follow a relapsing remitting (RR) disease course characterized by exacerbations (relapses) with periods of clinical stability in between. Patients may recover fully or partially from relapses (figure 3-top). On the opposite end of the spectrum, 15% experience a gradual worsening from onset with no initial exacerbations (primary progressive course, figure 3-bottom) Patients with an initial RR form of the disease may subsequently experience progression independently of relapse activity. This phenomenon is called secondary progression (SP, figure 3-middle). It is important to note that progressive disease (SPMS or PPMS) does not progress in a uniform fashion and patients may experience periods of relative clinical stability. Assessment of disease activity may provide insights into individual prognosis and guide patient management. Thus, current consensus recommendations have included disease activity as a modifier of basic phenotype courses mentioned above. Clinical activity is defined by relapses or radiological activity (presence of contrast-enhancing lesions, new or unequivocally enlarged T2 lesions).

Table 2 : Selection of neuroimaging studies performed within the last 5 years that have assessed the clinical relevance of cortical lesions using MRI.

 2

  to disability progression. Many studies have shown that GM volume correlates with measures of physical and cognitive disability in cross-sectional as well as longitudinal cohorts[147] [149] [150] [151] [152]. GM atrophy has been shown to be more useful than WM atrophy in prediction models of future disability and is therefore regarded as a valuable outcome measure in clinical drug trials[153].Unfortunately this measure is inherently non-specific and could be explained by the different processes taking place within the GM such as demyelination, glial pathology or neuronal damage. Postmortem studies have tried shedding light on the issue. Wegner and colleagues have shown that cortical thinning can occur within or outside lesions and is not related to the extent of cortical focal demyelination[105]. Recently,Popescu et al.

	Interestingly, GM atrophy doesn't follow the same dynamic pattern of accrual as WM
	atrophy. In a longitudinal study over 4 years, Fisher and colleagues demonstrated that
	GM atrophy increases with disease stage from CIS to SPMS while WM tissue loss
	remained constant [147]. Rocca et al. also reported that GM and WM damage followed
	different trajectories in the first 2 years after CIS [143], suggesting that these 2 events
	may be at least partly independent.

volume or cortical thinning

[142] [143] [144]

. Topographically, GM atrophy is most evident in the DGM structures such as the thalami, hippocampi, and in the cerebellum

[145]

[146] at the early stages of the disease. It later extends to neocortical structures and becomes widespread as MS progresses, clearly dominating the disease process in the later stages.

In CIS patients, GM volume loss can predict conversion to CDMS, and shorter time to second attack

[144] [148]

. GM volume has emerged as a clinically relevant measure with strong relation confirmed that in non-lesional neocortex, cortical volume was not related to myelin density

[107]

. This was later supported by another study using different sampling techniques

[154]

. These observations suggest that demyelination is not the primary substrate of volumetric loss and cortical thinning. In contrast, significant correlations were found with neuronal density, neuronal size and axonal density indicating that, at the later stages of the disease, MRI-measured GM atrophy is driven by the neurodegenerative process.

Table 3 : Demographic and clinical characteristics of healthy controls and MS patients included in the study (mean, range)

 3

Table 4 : Clinical measures of physical and cognitive performance for healthy controls and MS patients. * indicates a significant difference between the 2 groups.

 4

Table 5 : Clusters of decreased cortical [ 11 C]FMZ Bmax in MS patients compared to HC.

 5

Table 6 : Clinical correlations. Clinical performances of HC and MS patients and correlation with cortical [ 11 C]FMZ binding (partial correlation coefficient and p-value) adjusting for the effect of age and sex in the patient population (n=18).

 6

Area of between- group difference Side Cluster size (mm 2 ) Maxima Voxel MNI Coordinates CWP x y z

	Postcentral	L	3894.97	-18.1	-33.5	61.1	0.0002
	Posterior Cingulate	R	1586.47	6.4	-26.8	27.6	0.009
	Precentral	R	1894.19	27.3	-21.4	63.5	0.003

Table 7 : GLM clusters. Regions where cortical [ 11 C]FMZ Bmax in patients with multiple sclerosis (n=18) correlated positively with PASAT scores on surface based analyses. No negative correlation was found.

 7 FMZ Bmax, which estimates the density of GABAA receptors, showed more significant changes in the mildly atrophic RRMS subgroup compared to the most atrophic SPMS subjects. This was all the more unexpected as the influence of any partial volume effect should have resulted in more significant signal decrease in the grey matter of patients with marked atrophy. Therefore the impact of PVE on our results is probably minor, and this apparent discrepancy suggests that atrophy could induce an artificial increase in GABAA receptor density estimated by [ 11 C]FMZ Bmax. Such an artificial increase in density becomes easy to understand if we consider that the underlying substrate for grey matter atrophy is independent from the synaptic component[107].The pathological correlates of [ 11 C]FMZ binding changes detected in MS remain to be elucidated and may not be univocal. GABAA receptors are widely expressed by CNS neurons, both inhibitory and excitatory, and are enriched at axo-synaptic contacts[229]. Therefore quantification of [ 11 C]FMZ binding, while assessing mainly inhibitory neurotransmission, is not an intrinsic measure of GABAergic neurons themselves. The authors have shown that neurodegenerative processes drive GM atrophy in the cortex, as they found significant correlations between cortical volume and neuronal density or neuronal numbers, but no correlations with myelin density. In the present study, cortical thinning was significant in the temporal and occipital lobes bilaterally, reaching about 4-5% in ROI analyses. We related cortical thickness to [ 11 C]-FMZ binding, as a potential marker for neuronal integrity in vivo. FMZ binding across subjects in the MS group and the healthy control group, no correlation was found. These results do not antagonize the recent findings cited above. It can be postulated that [ 11 C]FMZ PET metrics reflect not only neuronal loss, but also subtle neurodegenerative changes such as synaptic loss or dendritic transections which may precede neuronal loss and are not fully captured by measures of GM volume. Therefore measuring neuronal damage by PET and GM volume by MRI does not provide redundant information and the combined use of

	1. IN VIVO QUANTIFICATION OF NEURONAL
	DAMAGE IN MS
	This work aimed to quantify the neuronal contribution to GM damage in MS using high
	resolution PET and the neuronal marker [ 11 C]FMZ. It is, to our knowledge, the first
	controlled in vivo study demonstrating changes in GABAA receptor density in MS
	patients. We found, using both a ROI and a surface-based approach, that cortical
	[ 11 C]FMZ binding was reduced in MS subjects, and that surface-based analyses allowed
	mapping of cortical damage at the group level. In the subcortical GM, a significant
	decrease in GABAA receptor sites but not density was detected in the thalami,
	hippocampi and amygdalae. Subgroup analyses suggested that [ 11 C]FMZ binding
	reduction was already present in RRMS subjects, whereas GM atrophy was barely
	detected at this stage. Finally, cortical GABAA receptor content was found to correlate
	positively with cognitive performance, suggesting the clinical relevance of this
	quantitative measure.
	MS is a diffuse chronic demyelinating disease of the CNS affecting not exclusively
	neurons, but also macroglial, microglial and inflammatory cells. In order to detect
	neuronal damage that could occur randomly in the GM of MS subjects, we took
	advantage of recent methodological optimizations. First, all PET images were acquired
	on a high-resolution research tomograph (HRRT, Annex B) which has one of the highest
	sensitivity and spatial resolution among currently available PET scanners (2.5 mm) and
	allows accurate localization of alterations in GABAA receptor density within the cortical
	ribbon. Secondly, we used the partial saturation method [216] to estimate receptor
	concentration from a single FMZ injection and without arterial blood sampling. This
	previously validated method exploits the natural decrease of bound ligand concentration
	over the duration of the experiment after labeled/unlabeled FMZ co-injection and
	provides an absolute quantification of BZD receptors concentration (Bmax). Thirdly, we
	supplemented ROI-based analyses with a surface-based approach to regionally map the
	differences in cortical [ 11 C]FMZ binding between MS patients and controls. While ROI
	analyses are constrained to predetermined GM regions either manually or automatically

Relationship between WM and GM damage

  The mechanisms underlying structural and functional neuronal damage within the MS grey matter haven't yet been elucidated, especially its relation to demyelination and inflammation. Of particular interest is the association between neurodegeneration and focal white matter damage, the most striking feature of the disease. Our group of patients presented a wide range of T2 lesion burden. Our results showed a significant yet moderate relationship between WM T2-weighted lesion load and the cortical GABAA receptors. The relationship was stronger in the subcortical GM. Our results support the notion that focal damage to WM tracts contribute at least partly to the on-going damage to neuronal and synaptic structures in the MS GM. This might be explained by highly connected structures such as the cingulate gyrus or the insula appear more prone to volume loss, reinforcing the role of WM disconnection in the development of GM atrophy. WM lesions, however only seem to account for less than half of the variance in GM atrophy in the early stages of the disease [258] and histopathological studies of late MS patients have outlined a dissocation between plaque load and pathological markers of neurodegeneration[259] [112]. These findings suggest the impact of other processes in the progression of GM and specifically neuronal damage.More subtle and diffuse changes occurring within the NAWM may also contribute to the cortical abnormalities observed using [ 11 C]FMZ PET. Such relationship was not evaluated in our pilot study. Quantitative MRI metrics have been shown to be sensitive to microstructural changes in post-mortem tissue [260] [112] and have been used to quantify white matter integrity in MS. In a recent paper, Kolasinski et al. also suggested that functional anatomical connectivity might be relevant to the spread of MS pathology in a "tract-specific pattern" [112]. Focal changes in MRI cortical thickness showed a clear intra-tract correlation with changes in WM microstructure. The correlation was reproduced using traditional histological metrics or DTI-derived measures of WM integrity. Tract-specific measures of WM integrity were not only found to better

	RRMS patients (n=9, age= 35.1 years, mean disease duration=23 months) and controls
	(n=7, age=32.3 years) is available. In a preliminary work (unpublished data), we related
	T2 lesion load and NAWM FA within 3 WM tracts (thalamo-prefrontal, cingulate and
	cortico-spinal) to [ 11 C]FMZ Bmax in anatomically related cortical areas (rostral middle
	frontal, posterior cingulate, precentral) in patients only. The cross-sectional correlations
	between these metrics failed to show significant relationship between focal or diffuse
	intra-tract WM damage and GM neuronal injury (figure
	underlying mechanisms of anterograde or retrograde degeneration [85]. Consistent with
	this notion that WM and GM are inter-related processes, several MRI studies have reported significant correlation between WM damage and GM atrophy, in deep GM structures [252] or cortical structures [253]. Longitudinal studies analyzing GM atrophy using VBM or similar techniques [254] [255] showed an association between global WM lesion volumes and increasing GM volume loss especially in fronto-temporal areas. Bernfeldt et al. demonstrated more widespread cortical GM atrophy in patients with longitudinal changes in T2 lesion volumes compared to patients with stable lesion loads [255] [256]. Location of WM damage was shown to contribute to clinical disability [238]. Similarly, the spatial distribution of WM T2 lesions is relevant to GM volume loss. Mulhau et al recently unveiled a significant spatial relationship between WM lesions and thalamus pathology [257]. This might be further explained by the fact that the thalamus is a relay organ, receiving input from multiple connected WM fibers. In the disability [179] than measures of global WM changes. Longitudinal study design could help understand the temporality of these events and determine whether tract-specific WM damage predicts GM as suggested by prior MRI work [253] In a new prospective project (clinicaltrial.gov ID: NCT01651520), we are planning to assess the chronology of occurrence and the prognostic value of neuronal damage measured by [ 11 C]FMZ PET in early RRMS and PPMS patients. Acquisition of multimodal MRI data at each timepoint will also help address the relationship between GM and WM pathology. Four groups of subjects will be included: i) patients with RRMS with less than 5 years disease duration (revised Mc Donald criteria, n=20); ii) patients with RRMS with over 5 years but less than 10 years disease duration (n=20); iii) MS cortex, correlate with corresponding cortical thickness but also related better to measures of patients with a primary progressive MS (PPMS) with less than 10 years disease duration
	(n=20); iv) healthy volunteers matched for age and sex (2/3 matched with RRMS
	patients; 1/3 matched with PPMS patients). Preliminary data of a subgroup of early
	105

Table 8 : Clusters of decreased CBF in MS patients compared to controls detected by surface based analyses.

 8

Acknowledgements

Comparisons of demographic and clinical variables were done using the Mann-Whitney U test for quantitative data.

For volume of interest analysis, the following metrics were analyzed:

-Volume normalized by head size (volume fraction) Comparisons between MS patients and healthy controls were performed using linear regression analyses adjusting for the effect of age and sex with a significance threshold of p<0.05. Exploratory subgroup comparisons of imaging data between RRMS, SPMS and control subjects were performed using the Mann-Whitney U test.

Surface based comparisons were performed using a general linear model (GLM) to test Bmax signal differences between control and MS patients on a vertex-by-vertex basis, adjusting for age and sex. A statistical threshold of p<0.05 was used, corrected for multiple comparisons using a Z Monte-Carlo Simulation, with 5000 iterations and a vertex-wise threshold of 0.05. A similar method was used to compare the cortical thickness between controls and patients across the whole cortex.

Subgroup analyses of [ 11 C]FMZ binding

Two groups of patients were included in our study: RRMS (n=9) and SPMS (n=9).

Considering the small sample size of these groups, such analyses are only exploratory but can provide some interesting information regarding the chronology of [

Publications

Related to doctoral work:

Oral communications

Regional:

Auteur : Léorah Freeman Directeur : Pr Bruno Stankoff Titre de la thèse en Français :

Imagerie de la dégénérescence neuronale dans une maladie démyélinisante: la sclérose en plaques.

Résumé :

La sclérose en plaques (SEP) a longtemps été considérée comme une affection inflammatoire démyélinisante de la substance blanche. (SB) Hors, de nombreuses études ont démontré l'implication extensive de la substance grise (SG