Abstract WP362: Diabetes Enhances Fibrotic Responses After Ischemic Stroke: Insights from Single-cell and Spatial Transcriptomics

Introduction: Diabetes mellitus (DM) is associated with elevated inflammation and poorer outcomes after ischemic stroke. During the sub-acute phase of stroke, glial and fibrotic scars began to form around the necrotic core; however, excessive or prolonged scarring can interfere with neural repair and functional recovery. Brain microglia and monocyte-derived macrophages (MdMs) are known to drive both inflammation and tissue remodeling, although it remains unclear how these cell types and processes are affected by DM. Methods: Single-cell RNA sequencing (scRNAseq) and Xenium-based highplex in situ hybridization were employed to identify stroke or diabetic effect on brain inflammation and gene expression in db/db and db/+ mice underwent distal middle cerebral artery occlusion (dMCAO) or sham surgery. Based on the differentially expressed genes identified in scRNA-seq, a 297-gene panel was constructed for the Xenium experiment to map spatial gene expression at both 3 and 14 days post-stroke. Immunofluorescence (IF) staining was performed to validate changes in fibroblasts and profibrotic markers. Results: In scRNAseq, a cluster previously associated with MdMs showed higher expression of fibrosis-related genes (e.g., Col4a2, Igfbp7 ) and lower expression of MHC class II genes ( H2-Ab1, H2-Aa ) in db/db compared to db/+ brains. Several subtypes of collagen genes and profibrotic gene Tgfb1 were among the top ranked ligands predicted by NicheNet. Xenium corroborated an increased expression of fibrotic markers (e.g., Col1a1, Col1a2, Fn1, Lum ) at day 14, in contrast to reduced MHC class II-expressing cells in stroke lesion comparing db/db to db/+ brains. IF confirmed a greater fibrotic deposition at 3-14 days after stroke in diabetic compared to non-diabetic controls. Conclusions: Integration of scRNAseq and Xenium produced a comprehensive atlas of post-ischemic cell types, their transcriptional features and spatial localization, revealing enhanced fibrosis after stroke in the diabetic relative to control mice. Ongoing investigation will determine the mechanisms underlying the profibrotic signaling in the diabetic brain after stroke and assess the potential therapeutic effect of inhibiting fibrosis in improving stroke outcome and recovery.