Abstract DP134: Ischemic Demyelination Induced by Aged Erythrocytes

Introduction: White matter hyperintensities of aging (leukoaraiosis), frequently seen on MRI, have an etiology that remains obscure. The underlying neuropathology of these lesions includes prominent demyelination, thought to be due to ischemic injury. We studied the potential contribution of erythrocytes (RBC) to the pathogenesis of ischemic demyelination using a mouse model in which RBC are biochemically aged and rigidified by treatment with the oxidative stressor tert-butyl-hydroperoxide (t-BHP). Methods: Tie2-GFP mice were injected with t-BHP-treated RBC and compared with mice injected with PBS-treated RBC. After 4 hours or 24 hours circulation, mice were perfused with PBS and fixed with 4% paraformaldehyde. Brain samples were collected and processed for immunofluorescence and single-cell spatial transcriptomics. Adjacent sections were examined under confocal microscope to evaluate RBC attachments and then aligned with sections for analysis of transcriptomics. Aligning histology images with gene spatial maps allowed visualizing and analyzing gene expression related to RBC-endothelium interaction induced by t-BHP treatment. Cell types were annotated based on primary marker genes, and after processing, a total of 194,306 cells were retained. In separate experiments, immunostaining was performed for myelin basic protein and hypoxia-inducible factor-1 alpha (HIF-1α) at 24 hours (n=4 mice/group). Statistical analysis was performed using linear mixed effects model. Results: Significantly higher RBC attachments and stalls in capillaries were observed in t-BHP-RBC mice at 4 hours and 24 hours vs PBS-RBC mice. Analysis of differentially expressed genes showed significant (P<0.001) upregulation at 4 hours for endothelial intercellular adhesion molecule-1 (ICAM-1, an adhesive molecule for RBC) and platelet-endothelial cell adhesion molecule-1 (PECAM-1, known to be upregulated under conditions of low shear expected in RBC attachment/stalls) in t-BHP-RBC mice. At 24 hours, mean fluorescent intensity (MFI) for myelin basic protein was 15 ±1.5 vs 24 ± 3.1 (p<0.01) for t-BHP-RBC mice vs PBS-RBC mice, respectively, while MFI for HIF-1α was 13.5 ±1.2 vs 9.8 ±1 (p<0.05), respectively. Conclusion: Aged RBC produce upregulation of endothelial adhesion molecules, significant capillary stalling of RBC, and demyelination with hypoxia. These findings offer novel insights into the pathogenesis of ischemic demyelination.