Moccia, Marcello (2018) Post mortem and in vivo study of multiple sclerosis pathogenesis. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Lingua: English
Title: Post mortem and in vivo study of multiple sclerosis pathogenesis
Date: 27 November 2018
Number of Pages: 97
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Clinica e Chirurgia
Dottorato: Neuroscienze
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
Brescia Morra, VincenzoUNSPECIFIED
Date: 27 November 2018
Number of Pages: 97
Uncontrolled Keywords: neuroscience; neurology; multiple sclerosis; neuroimaging; pathogenesis.
Settori scientifico-disciplinari del MIUR: Area 06 - Scienze mediche > MED/26 - Neurologia
Date Deposited: 19 Dec 2018 11:15
Last Modified: 30 Jun 2020 09:06


Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system. A number of pathological mechanisms could be responsible for acute demyelination and chronic tissue remodelling in MS, including inflammation, oxidative stress, microglia activation, and astrocyte infiltrates. In the present work, we aim to further explore the heterogeneity of MS pathogenesis on post mortem brains, and to evaluate the possibility to study MS pathogenesis by using magnetic resonance imaging (MRI) and peripheral blood biomarkers. In the first part of the study, we applied a data driven approach to classify MS patients in relation to the variety of pathological changes occurring in lesional and normal-appearing (NA) white matter (WM) and grey matter (GM), with subsequent clinical correlates. Tissue blocks from 16 MS brains were immunostained and quantified for neuro-axonal structures (NF200), myelin (SMI94), macrophages (CD68), B-lymphocytes (CD20), T-lymphocytes (CD3), cytotoxic T-lymphocytes (CD8), microglia (IBA1), astrocytes (GFAP), and mitochondrial damage. After semi-automatic registration of digitized histologic sections, regions-of-interest (ROIs) were manually defined in lesion and NA WM and GM. A latent class analysis was employed to characterize pathology subtypes in MS; different goodness of fit parameters (AIC, BIC, and G2 statistics) were used to identify the number of classes that better characterize the MS sub-populations. Profile 1 (active remodelling) was characterized by normal-appearing neuro-axonal structures and intact energetic metabolism, with high levels of macrophages/microglia and astrocytes. Profile 2 (mitochondrial dysfunction) was characterized by severely impaired mitochondrial function, along with demyelination and neuroaxonal loss, and ongoing inflammatory changes, mainly driven by cytotoxic T-cells (CD8+); patients in profile 2 presented with more severe symptoms at onset and faster disability accrual, when compared with other profiles. Profile 3 (inactive) was characterized by severe demyelination and axonal loss, with similarly reduced mitochondrial function, without any concomitant pathological process contributing to further tissue remodelling and/or damage. The possibility to classify each patient depending on his/her prevalent pathology profile support the concept of MS immunopathological homogeneity within the same patient and heterogeneity between different patients, and could be used to better profile MS patients and individualize their treatment. In the second part of the study, we explored post mortem pathology-MRI correlates and specifically focused on an advanced MRI technique (magnetization transfer ratio -MTR-), ideally detecting myelin content. MTR is widely used in MS observational studies and clinical trials, but its pathological correlates remain unclear. MTR maps were acquired at 3 Tesla from sixteen fixed MS brains and four healthy controls. 101 tissue blocks were immunostained and quantified, as previously described. After semi-automatic registration of digitized histologic sections and MTR maps, ROIs were manually defined. Associations between MTR and each stain were explored using linear mixed regression models (with cassettes nested within patients); differences in the associations between ROIs were explored using interaction terms. Lower MTR was associated with lower levels of NF200, SMI94, CD68, IBA1 and GFAP, with higher levels of CD8 and greater mitochondrial damage; MTR was more strongly associated with SMI94 in GM than WM. In a multivariate linear mixed regression model including all ROIs and brains, SMI94 was the best correlate of MTR. Myelin immunostain intensity is the strongest correlate of MTR, especially when measured in the GM. However, the additional histological correlates of MTR have to be kept in mind when interpreting the results of MTR clinical studies and designing experimental trials in MS. Finally, we evaluated the possibility to study (and to modify) MS pathology in vivo, by using biomarkers in the peripheral blood. Considering that oxidative stress is a driver of MS pathology, we evaluated the effect of coenzyme Q10 (CoQ10) on laboratory markers of oxidative stress and inflammation, and on MS clinical severity, and, then, calculated the sample size needed to detect significant variations to define most promising biomarkers. We included 60 relapsing-remitting MS patients treated with Interferon-Beta1a-44μg with CoQ10 for 3 months, and with Interferon-Beta1a-44μg alone for 3 more months (open-label cross-over design). At baseline, 3- and 6-month visits, we measured markers of scavenging activity, oxidative damage and inflammation in the peripheral blood, and collected data on disease severity. After 3 months, CoQ10 supplementation was associated with improved scavenging activity (as mediated by uric acid), reduced intracellular reactive oxygen species production, reduced oxidative DNA damage, and shift towards a more anti-inflammatory milieu in the peripheral blood (with higher IL-4 and IL-13, and lower Eotaxin, GM-CSF, HGF, IFN-γ, IL-1α, IL-2R, IL-9, IL-17F, MIP-1α, RANTES, TNF-α and VEGF). Also, CoQ10 supplementation was associated with lower expanded disability status scale, fatigue severity scale, Beck's depression inventory, and visual analogic scale for pain. For sample size estimates, we used adjusted-beta-coefficients of observed 3-month variation for each laboratory measure (and respective standard deviation); we assumed that the observed variation was the highest achievable treatment effect (100%), and we estimated sample size for conservative treatment effects (e.g., 70%), smaller than what observed. Setting 5% alpha-error and 80% power, low sample size requirements to detect 70% observed variation from a baseline pre-treatment timepoint to a 3-month follow-up were found for IL-3 (n=1), IL-5 (n=1), IL-7 (n=4), IL-2R (n=4), IL-13 (n=6), IL-6 (n=14), IL-8 (n=22), IL-4 (n=23), RANTES (n=25), TNF-α (n=26), IL-1β (n=27), and uric acid (n=29). CoQ10 supplementation improved scavenging activity, reduced oxidative damage, and induced a shift towards a more anti-inflammatory milieu, in the peripheral blood of relapsing-remitting MS patients treated with Interferon-Beta1a 44μg, along with clinical improvements. Peripheral biomarkers of oxidative stress and inflammation could be used in small proof-of-concept studies to quickly screen the mechanisms of action of new or already-existing medications for MS.


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