Abstract WP005: CSF-Delivered miR-204-5p Enhances Post-Stroke Recovery via PI3K/Akt-Mediated Astrocyte Modulation

(Background): Stroke remains one of the leading causes of death and long-term disability globally. Despite advances in acute care, effective treatments for the chronic phase of ischemic stroke are still lacking, aside from rehabilitation. Recent studies have shown that young cerebrospinal fluid (CSF) can restore oligodendrogenesis and improve memory function in aged mice. However, whether specific components within CSF contribute to functional recovery during the chronic stage of ischemic stroke has not been elucidated. In this study, we aimed to identify a specific microRNA within CSF and explore its potential therapeutic effects. (Method): Rats (n=47) underwent permanent left middle cerebral artery occlusion (pMCAO). CSF and brain samples were collected at 3, 7, 14, and 28 days post-pMCAO. MicroRNA expression in CSF was analyzed by array, and a target microRNA was selected based on heatmap and volcano plot. Proteomic analysis identified associated signaling pathways. A mimic or inhibitor of the microRNA was applied to primary astrocytes and neurons after 3-hour oxygen-glucose deprivation (OGD), and protein expression was evaluated at 96 hours (n=14). In vivo, the mimic was administered into CSF via cisterna magna at day 7. Behavioral tests and histological analyses were performed up to 28 days post-pMCAO. (Result): miR-204-5p was significantly upregulated in the CSF at 7 days after pMCAO and was selected as the target microRNA. In post-OGD astrocytes, application of a miR-204-5p mimic significantly reduced GFAP expression. Proteomic analysis suggested that miR-204-5p is associated with the PI3K/Akt signaling pathway. Consistently, treatment with the PI3K/Akt inhibitor LY294002 produced similar reductions in GFAP expression. In post-OGD neurons, the miR-204-5p mimic tended to increase the expression of MAP2, pNFH, pStat3, and Bcl-2. In vivo, rats receiving intrathecal administration of the miR-204-5p mimic exhibited improved functional outcomes and reduced infarct volumes at 28 days after pMCAO. Immunofluorescence analysis of the peri-infarct area revealed a significant decrease in astrocytes and a corresponding increase in neurons in the mimic-treated group compared to vehicle. (Conclusion): Intrathecal administration of a miR-204-5p mimic promoted functional recovery by suppressing astrocyte activation through the PI3K/Akt signaling pathway. This approach holds promise as a potential exosome- or liposome-based therapeutic strategy for future clinical applications.