007 – A bioinformatics approach to study microglial morphology and surveillance in CNS diseases
Ilias Roufagalas (1) – Maria Avloniti (1) – Lesley Probert (1) – Vasiliki Kyrargyri (1)
Hellenic Pasteur Institute, Laboratory Of Molecular Genetics, Athens, Greece (1)
Microglia, the brain immune cells, are highly ramified cells that continuously extend and retract their processes to survey the healthy brain and contribute to tissue homeostasis. In response to injury, infection and disease they change their morphology becoming hypertrophic and less ramified, they express different genetic profiles and they can have both beneficial and detrimental roles, depending on the nature and strength of the activating stimuli. Neuroinflammation and demyelination, which are hallmarks of Multiple Sclerosis (MS), both implicate microglia functions and gene alterations. Although microglia have been extensively studied for their role in MS, a high-throughput spatiotemporal analysis of their morphology and motility in the brain during the progression of the disease has not been described so far. In this study we developed a bioinformatics approach to study how microglia morphology and motility are affected in the brain of mice in two well-established models for MS, Experimental Autoimmune Encephalomyelitis (EAE) and Cuprizone-induced demyelination (CPZ). In a longitudinal study, C57BL/6 mice were sacrificed at different time-points of each disease, followed by perfusion/fixation, cryostat sectioning, Iba1 immunohistochemistry and confocal imaging of different brain areas. In parallel, motility analysis was performed on CX3CR1-GFP mice in the two models and at the same time-points, using brain craniotomy and intravital 2-photon imaging. For the analysis, confocal images and 2-photon videos were initially pre-processed with filter application and drift correction using custom written and existing plugins in Fiji, followed by an automated tracing method using Vaa3D and Neutube. Finally, a cell-based analysis was performed using a combination of imaging tools including Fiji and Imaris 3D and a task specific application developed in Java and MATLAB. The results so far show a strong correlation between demyelination and microglia responsiveness in the whole brain, while neuroinflammation affects microglia at more localised areas.
Supported by the Hellenic Foundation for Research & Innovation (Act 1156).