IMAGING NEURONAL DAMAGE IN THE MS GREY MATTER USING PET WITH [11C]FMZ 

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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 [40].
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. [21], 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% [41]. 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.

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 [43]. 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 [44].
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 [47].
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 [17]. 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 axon-myelin interactions [50]. 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 [51]. A patient may thus exhibit extensive or low remyelinating capacity. This was further confirmed by PET study of MS patients using radiotracer [11C]PIB, a thioflavine derivative sensitive to changes in myelin content in tissue (figure 7) [52]. We recently longitudinal follow-up of MS patients with [11C]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.

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, Brownell and Hughes (1962) [79] 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 [80], reaching up to 68% in extreme cases [58]. Grey matter demyelination is particularly extensive in the spinal cord, cerebellum, cingulate gyrus [80], thalamus [81] and hippocampus [82], 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 [82].
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%) [81], whereas subpial lesions are more frequent at the cortical level.

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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 [83]. 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. Wegner et al. quantified neuronal damage in the MS neocortex. The authors found a 10% reduction in mean neuronal density in leukocortical lesions compared to normally myelinated cortex, with decrease in neuronal size and significant changes in neuronal shape [105] [106]. Synaptic loss was significant in lesional cortex and occurred in greater proportion than neuronal soma reduction (-50% synaptophysin signal), suggesting that loss of dendritic arborization is an important feature in MS [105]. Pathologic changes in neuronal morphology, as well as reduced neuron size and axonal loss were also detected in normal-appearing cortex compared to controls [105] [107]. Klaver et al. reported a 25.4% loss of NeuN-positive neurons, reduced axon density (-31.4%) and 11.4% smaller neurons in type III cortical lesions (figure 12), while axonal density and neuronal size were also significantly reduced in the NAGM (-33.0% and -13.1% respectively) [108].

Proton Magnetic Resonance Spectroscopy

Proton Magnetic Resonance Spectroscopy (1H-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 1H-MRS either within small volumes (single voxel 1H-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.

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.

Table of contents :

LIST OF FIGURES
LIST OF TABLES
ABBREVIATIONS
INTRODUCTION
MULTIPLE SCLEROSIS: FROM INFLAMMATION TO NEURODEGENERATION
1. GENERAL CONSIDERATIONS 
1.1. EPIDEMIOLOGY
1.2. DIAGNOSIS OF MULTIPLE SCLEROSIS
1.3. EVOLUTION AND PROGNOSIS
2. MECHANISMS OF DISABILITY PROGRESSION 
2.1. WHITE MATTER DAMAGE
2.2. GREY MATTER INVOLVEMENT
IMAGING NEURODEGENERATION IN THE MS GREY MATTER
1. GREY MATTER ATROPHY 
2. QUANTITATIVE MRI 
2.1. DIFFUSION TENSOR IMAGING
2.2. MAGNETIZATION TRANSFER IMAGING (MTI)
2.3. PROTON MAGNETIC RESONANCE SPECTROSCOPY
3. POSITRON EMISSION TOMOGRAPHY 
3.1. PRINCIPLES OF PET IMAGING
3.2. [18F]-FLUORODEOXYGLUCOSE (FDG) PET AND NEURONAL METABOLISM
3.3. FLUMAZENIL, AN ANTAGONIST OF THE CENTRAL BENZODIAZEPINE RECEPTOR
IMAGING NEURONAL DAMAGE IN THE MS GREY MATTER USING PET WITH [11C]FMZ
1. SPECIFIC AIMS OF THE STUDY 
2. MATERIAL AND METHODS
2.1. SUBJECTS
2.2. CLINICAL EVALUATION
2.3. MAGNETIC RESONANCE IMAGING DATA ACQUISITION
2.4. PET ACQUISITION AND QUANTIFICATION
2.5. PROCESSING
2.6. STATISTICAL ANALYSES
3. RESULTS 
3.1. CHARACTERISTICS OF MS PATIENTS AND HEALTHY CONTROLS
3.2. [11C]FMZ BINDING IN THE GREY MATTER
3.3. GM ATROPHY IN MS PATIENTS
3.4. REDUCED [11C]FMZ BINDING IN THE MS GM
3.5. SUBGROUP ANALYSES OF [11C]FMZ BINDING
3.6. CORTICAL MAPPING OF [11C]FMZ BINDING CHANGES
3.7. RELATIONSHIP BETWEEN [11C]FMZ BINDING AND WHITE MATTER LESIONS LOAD
3.8. RELATIONSHIP BETWEEN [11C]FMZ BINDING AND CLINICAL METRICS
DISCUSSION & PERSPECTIVES
1. IN VIVO QUANTIFICATION OF NEURONAL DAMAGE IN MS 
2. CLINICAL RELEVANCE OF PET WITH [11C]FMZ 
3. UNDERLYING MECHANISMS OF NEURONAL DAMAGE IN MS
3.1. RELATIONSHIP BETWEEN WM AND GM DAMAGE
3.2. RELATIONSHIP BETWEEN GM DEMYELINATION AND NEURODEGENERATION
3.3. OTHER ASPECTS
4. FUTURE ROLE OF [11C]FMZ PET IN MS 
5. LIMITS OF PRESENT STUDY 
CONCLUSION
ANNEX 
1. ANNEX A
2. ANNEX B
3. ANNEX C

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