Fig 2: Rodent study demonstrates upregulation of Netrin-1 and its receptor UNC5a in microglia after ischemic stroke. A published database (GEO148350) provided RNAseq results on rat cerebral microglia in an MCAO model. We analyzed these data using GEO2R online bioinformatics tools with R language coding to extract individual values. We focused on microglia isolated from young rats (3 months of age) at day 3 after induction of ischemia and from sham treatment, which were most relevant to our study. (A) A boxplot. (B) A UMAP plot. (C) A mean-difference plot. (D) A volcano plot. (E) Array reads for genes related to microglia polarization and Netrin-1 receptor signaling. *p<0.05. NS, no significance.

Fig 3: Netrin-1 reduces apoptosis and invasiveness of microglia in vitro through UNC5a. (A) CCK-8 assay to assess the viable cells of pTMEM119-Netrin-1- and pTMEM119-Scr- transfected microglia in hypoxia condition, with/without presence of UNC5a-Fc. (B) Analysis of apoptotic cell death by Annexin V assay, shown by quantification (B) and by representative flow charts (C). (D) TUNEL staining shown by quantification and representative images. (E, F) Scratch cell migration assay, shown by representative images (E) and by quantification (F). *p<0.05. Scale bars were 50µm.

Fig 4: Acute ischemic stroke patients exhibit increased cerebral microglia expressing high Netrin-1 and UNC5a. (A) Immunostaining for TMEM119 in brain tissue from patients who had died from acute ischemic stroke (AIS), as well as from age-matched normal cases (NC). (B) Quantification of the number of TMEM119-postive microglia in the brain. (C–F) Microglia were isolated from AIS and NC brain tissue using flow cytometry, for which microglia were determined and sorted out based on their co-expression for TMEM119 and CD45, shown by representative flow charts (C), and by quantification (D). (E, F) RT-qPCR (E) and ELISA (F) for Netrin-1 and its main receptors, which include DCC, UNC5a, UNC5b, UNC5d and A2B, in isolated microglia. *p<0.05. Scale bars were 100µm.