The Role of Metals in Multiple Sclerosis Pathology

Background: Multiple sclerosis (MS) is a chronic disease of the central nervous system. Changes in iron, oxidative stress and dysregulation of metalloproteins in the serum, CSF, and brain tissues of MS patients, as well as excess extracellular iron accumulation have been proposed in MS pathogenesis. Aims: The objective of this proposal is to determine whether metal dyshomeostasis is associated with specific MS plaque types, immunopatterns and clinical characteristics or outcomes. Our first aim is to define the regional and cellular distribution of iron in white matter MS lesions relative to age and topographically matched normal controls with respect to plaque type, structural features and immunopattern. We will define the anatomical and cellular distribution of other biometals able to induce radical formation and determine their relationship to iron and metalloproteins involved in defense against oxidative stress. We will also define the pattern of regional and cellular distribution of biometals (iron, copper, and zinc) in relation to age, gender, disease duration, course and disability. Methods: We will utilize a multidisciplinary approach that includes synchrotron rapid scanning X-ray fluorescence imaging, histological staining, immunohistochemistry and in situ hybridization to investigate metal heterogeneity in a unique sample of patients with a focus on the early MS stage. We will characterize MS plaques and evaluate immunopattern classification. Parenchymal inflammatory infiltrates are determined throughout all areas for a given demyelinating stage, based on topographical maps. Acute axonal injury is also determined in all stages of demyelinating activity. We will use synchrotron rapid scanning X-ray fluorescence (RS-XRF) and microfocused X-ray fluorescence (XRF) mapping to generate low and high resolution iron maps corresponding to the histopathologically characterized lesional maps. XRF reliably and quantitatively detects all chemical forms of iron, thus surpassing the inherent limitations of conventional single metal staining methods. The capability to see how dysregulation of iron affects other metals in adjacent tissues may prove the key to understanding global metal regulatory pathways in MS. Statisticians are involved in study design, data entry, and analysis from its inception. Statistical analyses will be based on linear mixed effects models fit with restricted maximum likelihood. Scientific impact: Our study will provide important insights regarding pathophysiological mechanisms responsible for brain metal homeostasis and excess metal accumulation in MS which is critical for elucidating and developing pharmacological interventions in MS. Our proposal will also facilitate future XRF-MRI correlative studies, paving the way to novel metal-based biomarkers to monitor disease activity and progression in neurodegenerative diseases.

Background and Aims: Multiple sclerosis (MS) is a chronic disease of the central nervous system. Changes in iron, oxidative stress and dysregulation of metalloproteins in the serum, CSF, and brain tissues of MS patients, as well as excess extracellular iron accumulation have been proposed in MS pathogenesis. The objective of this work is to determine whether metal dyshomeostasis is associated with specific MS plaque types, immunopatterns and clinical characteristics. Methods: We utilized a multidisciplinary approach that includes synchrotron X-ray fluorescence imaging, histological staining and immunohistochemistry to investigate metal heterogeneity in a unique sample of MS patients. The project was facilitated due to collaborations between the Medical University Vienna, the Mayo Clinic Rochester, USA, the University of Saskatchewan, Canada and the Stanford Synchrotron Radiation Lightsource (SSRL), USA. Results: First, we pathologically characterized available MS autopsy cases. Plaque type distribution was analyzed in relation to clinical data using multinomial regression models. We could show that disease duration, clinical course, age and gender contribute to the dynamic nature of MS pathology. Thus, an adequate sample for XRF analyses was selected. Pathological, topographical lesion maps of white and gray matter plaques as well as areas of normal appearing white and gray matter were created. Next, a workflow had to be established how to process and analyze this unique dataset of combined XRF, pathological and clinical data. Pixel based XRF data were imported into a statistical program and linked to clinical and pathological data. Statistical models were fit. Our data showed significant differences in metal distribution not only among different plaque types and areas but also among different stages of the disease. Interpretation: Our findings lead to the conclusion that metal dyshomeostasis seems to play a crucial role in MS pathology. A possible impact on MS treatment especially in the chronic stage of the disease has to be further evaluated.