Department of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China.
Stroke. 2012 Mar;43(3):838-43. doi: 10.1161/STROKEAHA.111.635235. Epub 2012 Jan 5.
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).
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.
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.
AAV-NT-1 induced NT-1 hyperexpression before tMCAO reduced infarct size, enhanced neovascularization, and improved long-term functional recovery.
轴突导向因子 1（Netrin-1，NT-1）可促进体外内皮细胞增殖和迁移，并在体内促进成年大脑的局灶性新生血管形成。本研究在小鼠体内探讨了 NT-1 过表达对短暂性大脑中动脉闭塞（transient middle cerebral artery occlusion，tMCAO）后局灶性血管生成和长期功能结局的影响。
构建携带 NT-1 基因（AAV-NT-1）或 GFP（AAV-GFP）的腺相关病毒载体，并分别注射到 93 只小鼠的脑内。7 天后进行 tMCAO，随后再灌注 7-28 天。tMCAO 后 7-28 天，通过组织学和行为学评估检测结果。tMCAO 后 28 天，采用同步辐射微血管造影和免疫组织化学评估小脑血管网络和血管生成情况。
Western blot 和免疫组织化学结果显示，在 tMCAO 当天，正常和缺血半球均实现了 NT-1 过表达。免疫荧光成像显示，NT-1 表达主要在神经元和星形胶质细胞中。与生理盐水或 AAV-GFP 处理组相比，NT-1 过表达组的缺血性梗死面积明显减小（P<0.01）。同样，与生理盐水或 AAV-GFP 处理组相比，AAV-NT-1 处理组的神经功能缺损得到了极大改善（P<0.05）。此外，与其他 2 组相比，AAV-NT-1 处理组的血管生成增加（P<0.05）。tMCAO 后 28 天，活体同步辐射微血管造影显示，AAV-NT-1 处理组的血管分支比其他组更多。
tMCAO 前 AAV-NT-1 诱导的 NT-1 过表达可减小梗死体积，增强新生血管形成，改善长期功能恢复。
