Abstract TMP18: Multiplexed Histopathological Characterization Of Cerebral Microbleeds In The Aging Brain

Background: Cerebral microbleeds (CMBs) are manifestations of age-related microangiopathies associated with an increased risk of hemorrhagic stroke and cognitive decline. The pathophysiology of this age-related breakdown of the neurovascular unit remain unclear. Understanding CMB cellular architecture is needed for gene, pathway, and mechanism analyses, to pave the way for biomarker and therapeutic development. Herein, we aim to systematically describe histopathological changes in the CMB lesional milieu. Methods: Eight CMBs, along with contralateral non-lesional tissue, were identified by gross appearance at autopsy by a neuropathologist. Samples were first stained with H&E, then Prussian blue for non-heme iron, to localize the lesion. This was followed by multiplex fluorescence staining of β-amyloid and individual cell types including astrocytes (GFAP + ), macrophages (CD163 + /Iba1 + ), microglia (CD163 - /Iba1 + ), B-cells (CD20 + ), T-cells (CD3 + ), and endothelium (CD31 + ). The cell types within the lesional milieu, defined as 200μm from the periphery of the CMB, were quantified using QuPath. Astrocyte and microglia morphology were assessed via fluorescence imaging. Results: One CMB contained dilated capillaries, 2 contained lipohyalinized arterioles, and 4 contained dystrophic arterioles due to amyloid angiopathy. Astrocytes and macrophages showed higher normalized cell counts in all CMB tissues compared with controls ( p<0.005 and p<0.05 , respectively). Astrocytes in 7 of 8 CMB milieux showed reactive morphological changes, including larger cell bodies with peripheral nuclei. Microglia had higher normalized cell counts in 4 CMB tissues, with senescence-associated amoeboid, as well as ramified, reactive morphologies. The lesional milieu also showed higher counts of B-cells in 7 CMB tissues and T-cells in 3 CMB tissues when compared with controls. Conclusions: We provide the first description of the cellular architecture of the CMB and the associated lesional milieu. Despite heterogeneity of disrupted vasculature, there are common features of glial reactive changes and inflammation in CMBs. These results offer a basis for additional study of the CMB transcriptome and individual roles of respective cell types in CMB pathology.