Netrin-1 Hyperexpression in Mouse Brain Promotes Angiogenesis and Long-Term Neurological Recovery After Transient Focal Ischemia

Abstract

Background and Purpose— Netrin-1 (NT-1) stimulates endothelial cell proliferation and migration in vitro and promotes focal neovascularization in the adult brain in vivo. This in vivo study in mice investigated the effect of NT-1 hyperexpression on focal angiogenesis and long-term functional outcome after transient middle cerebral artery occlusion (tMCAO). Methods— Adeno-associated viral vectors carrying either the NT-1 gene (AAV–NT-1) or GFP (AAV-GFP) were generated and injected into the brains of separate groups of 93 mice. Seven days later, tMCAO followed by 7–28 days of reperfusion were carried out. Histological outcomes and behavioral deficits were quantified 7–28 days after tMCAO. Small cerebral vessel network and angiogenesis were assessed 28 days after tMCAO, using synchrotron radiation microangiography and immunohistochemistry. Results— Western blot and immunohistochemistry showed that on the day of tMCAO, NT-1 hyperexpression had been achieved in both normal and ischemic hemispheres. Immunofluorescence imaging showed that NT-1 expression was primarily in neurons and astrocytes. Ischemia-induced infarction in the NT-1 hyperexpression group was attenuated in comparison to saline or AAV-GFP–treated groups ( P <0.01). Similarly, neurological deficits were greatly improved in AAV–NT-1–treated mice compared with mice in saline or AAV-GFP–treated groups ( P <0.05). In addition, angiogenesis was increased in AAV–NT-1–treated mice compared with the other 2 groups ( P <0.05). In vivo synchrotron radiation microangiography 28 days after tMCAO revealed more branches in AAV–NT-1–treated mice than in other groups. Conclusions— AAV–NT-1 induced NT-1 hyperexpression before tMCAO reduced infarct size, enhanced neovascularization, and improved long-term functional recovery.