Effect of VEGF-C treatment on sorted brain microglia/macrophages

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intra-cerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth, and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon ligation of the dCLN afferent lymphatics, and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke. For evaluating the effect of VEGFc-mediated meningeal lymphatic expansion on brain myeloid cells, we treated C57/Bl6J mice with AAV-CTRL or AAV-VEGFc for 4 weeks. Half of the samples were obtained at this time point. For the other half of samples, tMCAO was performed and the infarcted hemisphere was isolated 7 days after. Samples were processed with MACS bead-based myelin removal followed by purification of CD11b+ microglia/macrophages using MACS beads. Isolated cells were then lysed for mRNA isolation and sequencing. We then performed gene expression analysis with RNAseq data.