Objective Netrin-1 is one of the axon-guiding molecules that are critieal for neuronal development.Netrin-1 was found to stimulate the proliferation and migration of human cerebral endothelial cells in vitro and also promote focal neovascularization in normal adult mouse brain in vivo.In the present study, we investigate the role of overexpression of netrin-1 via adeno-associated virus (AAV) mediated-gene transfer in a transient middle cerebral artery occlusion (tMCAO) model of mice.Methods We constructed adeno-associated virus carrying Netrin-1 (AAV-NT-1) or GFP gene (AAV-GFP, as a control).2.5 μL of AAV-NT-1 suspension with 2 × 109 virus particles was injected stereotactically into the lateral ventricle of male CD-1 mice at a rate of 0.2 μL/min.Same amount of AAV-GFP or saline was injected as controls (n=21 per group).Seven days after AAV-NT-1 injection, these mice underwent 60 min of tMCAO followed by 7 to 28 d of reperfusion.Netrin-1 expression was quantified by Western blot and Netrin-1 localization was determined by immunostaining.The infarct volume and animal behavioral outcomes (beam walk test and Rotor-Rod test) were examined until the end of experiment.Lectin-stained microvessels were counted to determine the effect of Netrin-1 on angiogenesis after tMCAO.Results Endogenous Netrin-1 expression was induced in the ischemic penumbra of mouse brain by transient MCAO.The expression of Netrin-1 in the ischemic hemisphere was further enhanced after AAV-NT-1 gene transfer.Both neurons and astrocytes but not endothelial cells expressed Netrin-1 following AAV-NT-1 gene transfer.Overexpression of Netrin-1 promoted the recovery of focal cerebra blood flow after MCAO in the AAV-NT-1-transduced mice compared to control group (P < 0.05).Brain infarct volume was decreased and neurobehavioral outcomes were greatly improved in the AAV-NT-1 transduced mice compared to the AAV-GFP or saline injected group of mice up to 28 days after tMCAO (P < 0.05).Microvessel counts were significantly increased in the left hemisphere of AAV-NT-1 transduced mice compared to the control group (P < 0.05).Conclusion Our results demonstrate that NT-1 not only plays a key role in neuronal development as reported before, but also participates in neuroprotection and neovascularization and vessel remodeling in the ischemic brain.