Cerebral microbleeds (CMBs) are age-related vascular lesions, predisposing patients to a lifetime risk of stroke and cognitive decline. CMBs most commonly occur in the presence of hypertensive vasculopathy or cerebral amyloid angiopathy. Little is known about the cellular architecture of the CMB lesional milieu as well as CMB pathobiological mechanisms. Understanding common histopathologic and transcriptomic signatures across heterogenous CMBs can aid in biomarker feature selection.

These knowledge gaps were first addressed using human CMB autopsy tissue and CMB tissue from an E4FAD mouse model, through immunohistochemical staining and quantification. Changes in counts of brain resident (astrocytes and microglia) and circulating inflammatory cells (macrophages, B-cells, and T-cells) were measured. Morphological assessment, and reactive marker staining, was then completed for astrocytes and microglia. A U-Net model was developed to measure reactive marker staining. Heterogeneities in human CMB vascular disruption included lipohyalinized arterioles, amyloid angiopathy-induced dystrophic arterioles, and dilated capillaries, with minimal variation in vascular dysmorphisms of murine CMB arterioles. Increased normalized counts of astrocytes and macrophages were identified surrounding the human CMB lesion, while there were increases in astrocytes and microglia in the murine lesions when compared to contralateral brain tissue (p<0.05). Both cell-types exhibited hyperreactive changes as well as slight increases in reactive marker staining surrounding CMB lesions. Common CMB histopathologic signatures, with respect to astrocytes, microglia, and macrophages, regardless of etiology or sex, justify their study as a single pathology.

Following cell-type characterization, assessment of lesional differentially expressed genes (DEGs) and mechanisms was completed. Spatial transcriptomics identified 781 DEGs in human CMBs as well as 448 DEGs in mouse CMBs, with 47 common DEGs (p<0.05, FDR-corrected). Pathway enrichment identified 110 common pathways related to vascular permeability, endothelial dysfunction, senescence, and inflammation (p<0.05, FDR-corrected). Lobar and deep CMBs, as well as CMBs in males and females, identified similarly enriched pathways. While etiology and sex-specific pathways implicate certain differential mechanisms, commonly enriched pathways further substantiate their study as a single pathology.

Spatial transcriptomics, through ortholog protein and miRNA-mRNA interactions, then informed of biologically plausible circulating biomarker candidates. Circulating miRNA candidates were identified through unsupervised miRNome sequencing of plasma from human CMB patients and E4FAD mice (p<0.1, FDR-corrected). Two miRNAs were discovered to be homologous between human CMB patients and E4FAD mice with mechanistic ties to IPA pathways enriched from the respective transcriptomes. A supervised literature search was then completed to identify 10 blood circulating proteins that had previously been assessed in CMB patients and healthy controls. Protein levels were assessed, and elastic net feature selection identified 7 circulating protein biomarker candidates.

This is the first report to characterize the CMB microenvironment, provide mechanistic insight into its pathobiology, and identify biologically plausible circulating diagnostic biomarker features. Future studies should identify mechanisms specifically related to lesion genesis, maturation, and progression to hemorrhagic stroke. In addition, studies aimed at validation of these features, and model validation in large multi-site cohorts are needed. The framework applied here paves the way for biomarker discovery in CMBs as well as other cerebrovascular diseases across varying contexts of use.