Abstract 21: Similarity in the Changes of Brain Myeloid Transcriptome Signature is Shared Between Ischemic Stroke and Chronic Inflammation Caused by Diabetes Mellitus

Abstract

Introduction: Ischemic stroke (IS) is a pressing global health concern. Diabetes mellitus not only heightens the risk of IS but also aggravates its outcomes. There is established evidence that neutrophil infiltration is involved in inflammation after stroke through various mechanisms, such as activating neutrophil extracellular traps. We aimed to examine the factors that contribute to the deterioration of IS in diabetic mice focusing on mechanisms underlying immune cell infiltration by single cell RNA sequencing (scRNAseq) of brain before and after stroke. Methods: Experimental stroke was induced using the distal middle cerebral artery occlusion (dMCAO) model. db/db mice were used to model type 2 diabetes and obesity, while db/+ mice served as euglycemic controls. Mononuclear cells from ipsilateral mouse brains were isolated 3 days post-dMCAO or sham surgery with the Percoll gradient following enzymatic digestion. Transcriptomic analysis was performed using scRNAseq from db/+ sham, db/db sham, db/+ stroke, and db/db stroke. Fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) staining were conducted to validate the changes detected in the transcriptome results. Results: Prior to the onset of stroke, db/db mice exhibited changes in gene expression in endothelial cells and myeloid cells including macrophages, monocytes, and microglia, mirroring patterns of changes after stroke. Among the macrophage subpopulation in particular, db/db sham and db/+ stroke mice shared an overlap of 638/901 (70.8%) differentially expressed genes compared to db/+ sham. Gene enrichment analysis indicated predominant upregulation of genes linked to leukocyte migration and neutrophil chemotaxis. FACS revealed a heightened neutrophil presence in the brain parenchyma of diabetic mice before stroke, and further elevated after stroke compared to control mice. Specifically, the increased expression of anaphylatoxin receptor C3aR and complement C3 shown by IF corroborated with the transcriptomic signature, supporting a role of complement activation and myeloid cell transmigration. Conclusions: Our findings provide valuable insights into the mechanisms whereby diabetes potentially escalates neutrophil infiltration post-stroke.