Perivascular Macrophages in ischaemic endothelial function

Kelk, Joe (2023). Perivascular Macrophages in ischaemic endothelial function. PhD thesis The Open University.



The word “stroke” is a universally understood concept that echoes throughout the world due to their regular occurrence, usually with dire consequences. To this date strokes remain the second leading cause of death worldwide and the single biggest cause of disability on our planet. Resulting in millions of deaths per year and debilitating side effects among survivors, who will require care possibly for the rest of their lives.

A key group of cells known as brain resident myeloid cells have been investigated in ischaemic conditions designed to mirror the most common form of stroke, with this larger cell population demonstrating dramatic responses to ischaemia. A particular sub-population of brain resident myeloid cell known as the Perivascular Macrophage (PvM) exist at the border of the Blood Brain Barrier (BBB) and have demonstrated ability to regulate endothelial permeability and the BBB as a whole. However, despite this PvMs have been largely unexplored and their responses in stroke remain largely unknown.

The aim of this project is to investigate how PvMs response in ischaemic conditions in both in vitro & in vivo models. Initial investigation will focus heavily on murine models of ischaemia at 24 hour, 48 hour and 4 day times points with (transient occlusion) or without (permanent occlusion) reperfusion. Using a combination of immunofluorescence and confocal microscopy, areas such as PvM density within lesions PvM morphology will be investigated. Structure Illumination Microscopy (SIM) will then be used to investigate the functional impacts of these changes, examining primary lysosomal activity within PvMs demonstrating phagocytosis and a key response of macrophages undergoing an inflammatory response.

Once this model and responses of PvMs has been established within a murine model, an emphasis will be placed on translational research, adapting these findings to a human condition. To replicate this human induced pluripotent stem cells (iPSCs) will be investigated to generate a macrophage cell type with similar characteristics to a tissue resident macrophage 3 similar to a PvM. This will involve creating and refining a custom differentiation protocol to produce the target cell type and confirming key tissue resident macrophage markers via immunofluorescence and qPCR.

Once the target cell has been derived the functional characteristics investigated previously in murine models will be investigated to determine if the iPSC derived cells can undergo the same inflammatory process of phagocytosis using the same SIM method. To further utilise these cells, more complex models will then be created, using 3D techniques to give rise to human cellular models suitable for study.

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