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Myeloarchitecture is a prominent feature that can identify the primary motor and sensory areas in the cerebral cortex and is increasingly imaged in magnetic resonance imaging (MRI) studies of cortical parcellation in humans. However, MRI studies of cortical myeloarchitecture are technically difficult for two reasons: the cortex is only a few millimeters thick, and intracortical contrast due to myelin is much smaller than the overall anatomical contrast between cortical tissue and underlying white matter that is typically utilized in imaging. The research in this thesis thus presents specific MRI protocols to visualize intracortical myelin, image processing protocols to delineate the heavily myelinated cortex from the adjacent typical cortex and the application of these techniques in the precentral motor cortex to study morphology of the highly myelinated dorso-medial part, consisting of Brodmann area (BA) 4 and part of BA 6. Optimization of the MRI protocols involved determining the sequence parameters for a T1-weighted MRI sequence to obtain maximal intracortical contrast at 0.7 mm isotropic resolution in imaging time of 15 min, based on T1 differences between cortex that is myelinated (GMm) or unmyelinated (GM). As part of the optimization, T1 values were measured in the following brain tissues: GM, GMm and white matter (WM). The optimization was carried out by simulating the MRI signal for a 3D, magnetization prepared, gradient echo sequence, using the measured T1 values in the analytical signal equations. It was found that lengthening the time delay at the end of each inner phase encoding loop increased the intracortical contrast. The optimization of MRI protocols also included implementing techniques to reduce radio frequency field (B1) inhomogeneities. It was found that dividing the optimized, T1-weighted MRI with a predominantly proton density weighted image resulted in a ratio image with significantly reduced B1 inhomogeneities. The goal of the image processing protocols developed in this thesis was to visualize the variation of intracortical myelin across the precentral motor cortex and to delineate its well-myelinated dorso-medial part. The myeloarchitectonic feature that was selected to visualize the variation in intracortical myelination was the thickness of GMm in the deeper parts of the cortex relative to the cortical thickness, referred to as the proportional myelinated thickness (p). To measure p, the following processing steps were performed. The ratio image was segmented into four tissues: GM, GMm, WM and cerebrospinal fluid (CSF) using fuzzy C-means clustering technique. Using a level set approach, thickness of the cortex was determined as the distance between the outer boundaries of GM and WM and thickness of GMm or myelinated thickness (m) was determined as the distance between the outer boundaries of GMm and WM. The proportional myelinated thickness p, was calculated as follows: p= m/t. The well-myelinated dorso-medial part of the precentral cortex, referred to as Mm, was distinguishable from the adjacent cortex when the proportional myelinated thickness was projected on the outer cortical surface. The optimized MRI and image processing techniques developed in this thesis were used to investigate cortical plasticity in amputees. Two morphological features of the myeloarchitecture over Mm, the mean proportional myelinated thickness and area, were measured in four lower limb amputees and four matched controls. A comparison of these morphological features showed no statistically significant difference (p < 0.05) between the two groups. / Thesis / Doctor of Philosophy (PhD)

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The normal aging process in humans is characterized by a number of hallmark changes including decreased white matter volume in the brain and accompanying cognitive decline. This is in contrast to neurodegenerative aging processes which involve acute pathology which results in neuronal cell death. Studying non-degenerative normal aging in humans can be difficult because of the high prevalence of neurodegenerative diseases in the population and other potentially confounding effects. Rhesus monkeys are an excellent model organism for the study of normal aging, as their aging process has been demonstrated to involve diminished white matter volume, but they do not suffer from neurodegenerative diseases such as Alzheimer's. In this study we seek to quantify levels of myelin degradation using confocal microscopy in regions of interest where it has been previously demonstrated that loss of white matter integrity results in lower levels of cognitive function across different treatment groups including aging monkeys, calorie restricted monkeys, and controls for calorie restricted monkeys. These areas include prefrontal white matter which is vital to executive function, the hippocampus which is integral to memory consolidation and the learning process, and finally the anterior, middle, and posterior cingulum bundle. The cingulum bundle contains a diverse variety of projections between cortical and subcortical regions, including but not limited to projections to and from the cingulate cortex which has been demonstrated to be vital for emotional processing, the limbic system, and a wide spectrum of other functions. We aim to quantify white matter degradation in these regions by using immunofluorescent tagging for healthy myelin basic protein (MBP) and degraded myelin basic protein (dMBP) and by measuring the colocalization between the two. For prefrontal white matter and hippocampus, we did not find significant differences in myelin degradation across treatment groups. In the cingulum bundle, however, we did find a significant effect of treatment on overall myelin damage throughout the bundle, and in particular we determined that there was a significant difference in colocalization in the anterior cingulum bundle between aging monkeys and control calorie restricted monkeys. Analysis of behavioral testing data yielded surprising results as we were unable to find a strong correlation between our measure for myelin degradation, and level of cognitive impairment. Our results indicate that there are likely differences in regional vulnerability to age related myelin damage across different white matter regions of the brain, however we would like to expand on this study to gain a more accurate understanding of how loss of white matter volume is distributed through the brain and the impact that has on cognitive outcomes.

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Stroke is the main cause of adult motor disability. Nevertheless, recent meta-analyses show that the theoretical models conceived to explain the post-stroke brain reorganization are inaccurate and therefore misleading in laying the theoretical foundation for rehabilitation protocols. Mixed results are reported especially in works investigating the excitability properties of the stroke injured brain. Shedding light on the reasons that brought to such mixed results is the central topic of this doctoral thesis. In particular, this confounding evidence is here discussed and tackled in the light of recent works employing brain-state dependent stimulation protocols. These works have been of paramount importance, as they showed that the effects of non-invasive stimulation (TMS and/or rTMS) on the hand knob of the motor cortex depend on the instantaneous sensorimotor state. This local state is largely determined by the phase of the mu-alpha oscillations, with the negative peak representing a high excitability condition. Brain-state dependent results show that controlling for the local state at the moment of stimulation is crucial in order to reduce variability in studies investigating cortical excitability: an approach that has never been employed in stroke literature, so far. In this doctoral thesis, new evidence is provided on affected and unaffected hemispheres’ excitability properties depending on the local state at the moment of stimulation. This previously uncontrolled state-dependent variability is here proposed as one of the factors at the basis of the mixed results in stroke literature. Furthermore, the current models aimed at explaining post-stroke brain reorganization do not take into account factors that recent works suggest might contribute to stroke recovery. In fact, it is here suggested that: interhemispheric inhibition should not be interpreted as competition, structural reserve should be assessed also at the level of the corpus callosum, diaschisis processes should be taken into account and structural and functional connectivity patterns should be included in patients’ assessment. Finally, the excitability properties of the stroke brain have been often inferred comparing stroke patients’ with young healthy controls’. In this regard, it is here proposed that only healthy peers should be included in the control groups, as brain structural changes due to healthy aging have an impact on corticospinal excitability. The aforementioned functional and structural issues are addressed in the following chapters by means of different techniques (i.e. TMS-EEG, MRI, MEG). In particular, in Chapter 1, a new framework of post-stroke brain reorganization is proposed, in which previously over-looked factors are suggested to be essential in the understanding of the potential plastic changes following stroke. Specifically, a new account where interhemispheric inhibition is interpreted in terms of integration and not competition, is supported. Moreover, the proposed framework includes recent pieces of evidence suggesting that structural reserve should be evaluated in the individual patient not only at the level of the cortex, but also in the different sections of the callosum. Finally, it is proposed that structural damage is not static, but rather dynamic as it continues also after the stroke episode through dischiasis processes. In Chapter 2, new knowledge is provided on the different excitability properties of the two hemispheres of stroke patients. In this chapter, TMS-EEG data of stimulation on both the affected and unaffected motor cortex in severe chronic strokes are analysed with a brain-state dependent approach. For the first time, it is shown that the excitability properties of the affected and unaffected hemispheres differ as the local state at the moment of stimulation influences the two hemispheres’ response differently. In particular, the strong and simplified TMS-evoked response in the affected hemisphere, previously reported in severe patients, is shown to depend on a disruption of the differentiation between the high and low excitability states of the motor cortex, determined by the instantaneous phase of alpha oscillations. This low differentiation between excitability states in the affected hemisphere should be systematically investigated, as it could be a potential feature of patients who experience poor recovery. Furthermore, in Chapter 3, connectivity at the individual alpha peak is investigated in a big cohort of healthy participants, in a resting state MEG dataset. This work was implemented because alpha connectivity networks have been shown to predict stroke recovery. For this reason, there is a necessity to reliably assess connectivity at alpha before and after rehabilitation, as this could be informative on the efficacy of rehabilitation. Specifically, it is shown that using complementary phase-coherence metrics is more effective to estimate connectivity patterns at source level. This compound approach is proposed as a tool to better control the modulatory effects of rehabilitation stimulation protocols, in order to identify which are the changes in activity patterns that are potentially responsible for recovery. Finally, in Chapter 4 brain structural changes associated with healthy aging are investigated in a big cohort of participants aged between 18 and 90 years old, both in terms of cortical thinning and cortical myelin concentration loss. In particular, given recent evidence on the relationship between cortical myelin content and corticospinal excitability, it is shown that age-dependent myelin loss occurs mostly at the level of the premotor, motor and sensory cortices. These structural changes need to be taken into account when stroke patients are compared with controls. In fact, since stroke patients are often in their elderly, these age-related structural changes need to be controlled by including only age-matched healthy participants in control groups, as this is not often a fulfilled criterion in stroke studies. To conclude, this doctoral thesis proposes that the current models’ inaccuracy depends on 1) patients’ individual structural and functional factors that have not been taken into account in previous models of brain reorganization post-stroke (Chapter 1), 2) brain-state dependent variability in stimulation effects that have not been controlled for in stroke literature (Chapter 2), 3) a lack of a systematic method to assess the effects of stimulation rehabilitation protocols (Chapter 3) and 4) structural brain changes due to healthy aging, that affect also the stroke brain, and that are not taken into account when patients are compared with young controls in corticospinal excitability studies (Chapter 4). To the author’s knowledge, this is the first work aimed at explaining mixed results in stroke literature from different perspectives and using different neuroimaging techniques for functional and structural anomalies, exploiting recent brain-state dependent approaches for the analysis of stroke patients’ data.

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Myelin wird durch einem speziellen Membran von Oligodendrozyten im ZNS hergestellt. Diese mehrschichtige Struktur umhüllt Axonen mit ihner trophischen Unterstützung und erleichtert die schnelle Übertragung von elektrischen Signalen. Um die kurzfristigen Auswirkungen der Demyelinisierung zu untersuchen, die histologische Analyse in einem Maus-Modell wurde durchgeführt, wo die myelinisierende Oligodendrozyten wurden abgetragen durch die Expression von Diphtherie-Toxin-Rezeptor in reifen Oligodendrozyten und systemische Diphtherie-Toxin Injektionen. Wir beobachteten, dass so eine Abtragung in einer tödlichen Krankheit resultiert, wo Demyelinisierung der weißen Substanz Bahnen durch Mikroglia Aktivierung von der axonalen Schäden begleitet wurde. Wir haben gezeigt, dass dieses Modell daher auch für das Studium von der kurzfristigen Demyelinisierung-vermittelte axonale Schädigung und Myelin Abbau geeignet ist. Um die Auswirkungen der reversibel Demyelinisierungepisoden auf langfristige Bewegungsapparates Leistung und neuro-axonalen Integrität zu untersuchen, wurden Cuprizon-behandelten Tieren mit motorischen Sequenz (MOSS) überwachtet. Mit MOSS haben wir beobachtet, ob eine funktionelle Erholung erreicht und in langfristig erhalten konnte. Trotz die komplette scheinbare Erholung, die behandelte Tieren zeigten eine late-onset motorischen Beeinträchtigung und laufenden akuten axonalen Schädigung. Dieses Modell imitiert viele Aspekte der axonalen Pathologie bei chronisch progredienter MS und könnte daher bei der Untersuchung der Faktoren, die Initiierung, Aufrechterhaltung oder Kompensation axonalen Schädigunggenutzt werden. Schließlich, weil Myelin Neuroprotektionwahrscheinlich eine direkte Kommunikation zwischen Axonen und Oligodendrozyten beinhaltet/braucht , Proteomanalyse der Myelin-Fraktionen in axo glialen Regionen ist durchgeführt, um neue Kandidaten in axo-glialen Interaktion im Rahmen des Myelin Biogenese beteiligt sind zu finden. Zahlreiche funktionalle Assays wurden gegründet und verwenden, um identifizierten Kandidaten zu bewerten, um ihre Rolle in axoglial Kommunikation und Myelinbildung zu bestimmen. Wir haben festgestellt, dass einige Mitglieder der IgLON Familie binden beide Oligodendrozyten und Axone. Wir beobachteten, dass diese Proteine kein Effekt auf die Migration, Proliferation, Differenzierung von der Oligodendrozyte-Vorläuferzellen haben. Allerdings beobachteten wir, dass ein Mitglied, Ntm wirkt sich negativ auf die frühen Phasen der Myelinisierung.

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