Abstract TMP75: Histopathologic and Transcriptomic Characterizations of Cerebral Microbleeds in an E4FAD Murine Model

Abstract

Background: Cerebral microbleeds (CMBs) are age-related vascular lesions, predisposing patients to a lifetime risk of hemorrhagic stroke and cognitive decline. The cellular architecture in the lesions and their genetic alterations have not been elucidated, limiting biomarker development and therapeutic targeting. Herein, we report histopathological and transcriptomic signatures of CMBs in a murine model. Methods: Seven APOE4 + / + ; 5xFAD mice were sacrificed at 8 months and had their brains harvested. Brain tissue was stained with H&E and Prussian Blue for non-heme iron to identify CMBs. Astrocytes (GFAP + ), microglia (CD163 - /Iba1 + ), macrophages (CD163 + /Iba1 + ), B cells (CD20 + ), T cells (CD3 + ), and endothelium (CD31 + ) were stained using multiplexed immunofluorescence. Regions of interest (ROIs) around CMBs and directly adjacent tissue, as well as contralateral control brain tissue, were selected and quantified using QuPath. GeoMx spatial transcriptomics was performed on lesions and contralateral control brain areas in the same mice. The DEG analysis was completed using DESeq2. Results: CMBs had higher astrocyte and microglia counts (normalized to ROI area) than control tissue (p<0.05). Microglia showed reactive, amoeboid morphological changes: hypertrophic cell bodies and shorter processes. T cells had a trend toward higher cell counts in CMBs (p=0.12). Macrophages and B cell counts were similar between CMBs and controls. The spatial transcriptomics identified 19 DEGs between CMBs and control tissue (p<0.1, FDR corrected) related to permeability, cell metabolism, apoptosis, and transcriptional regulation. Conclusions: This is the first report relating the CMB lesional microenvironment to spatial transcriptomic analyses. Further studies shall aim at confirming these findings in human CMB lesions, and their association with circulating molecules. The results pave the way for enhanced mechanistic insight, biomarker discovery and potential therapeutic targeting.