Department of Neurology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China.
Neuroscience. 2013 Feb 12;231:225-32. doi: 10.1016/j.neuroscience.2012.11.059. Epub 2012 Dec 8.
Neurological deficit following cerebral infarction correlates with not only primary injury, but also secondary neuronal apoptosis in remote loci connected to the infarction. Netrin-1 is crucial for axonal guidance by interacting with its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5H (UNC5H). DCC and UNC5H are also dependence receptors inducing cell apoptosis when unbound by netrin-1. The present study is to investigate the role of netrin-1 and its receptors in ipsilateral ventroposterior thalamic nucleus (VPN) injury secondary to stroke in hypertensive rats. Renovascular hypertensive Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO). Continuous intracerebroventricular infusion of netrin-1 (600 ng/d for 7 days) or vehicle (IgG/Fc) was given 24h after MCAO. Neurological function was evaluated by postural reflex 8 and 14 days after MCAO. Then, immunoreactivity was determined in the ipsilateral VPN for NeuN, glial fibrillary acidic protein, netrin-1 and its receptors (DCC and UNC5H2), apoptosis was detected with Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) assay, and the expressions of caspase-3, netrin-1, DCC, and UNC5H2 were quantified by western blot analysis. MCAO resulted in the impaired postural reflex after 8 and 14 days, with decreased NeuN marked neurons and increased TUNEL-positive cells, as well as an up-regulation in the levels of cleaved caspase-3 and UNC5H2 protein in the ipsilateral VPN, without significant change in DCC or netrin-1 expression. By exogenous netrin-1 infusion, the number of neurons was increased in the ipsilateral VPN, and both TUNEL-positive cell number and caspase-3 protein level were reduced, while UNC5H2 expression remained unaffected, simultaneously, the impairment of postural reflex was improved. Taken together, the present study indicates that exogenous netrin-1 could rescue neuron loss by attenuating secondary apoptosis in the ipsilateral VPN after focal cerebral infarction, possibly via its receptor UNC5H2, suggesting that relative insufficiency of endogenous netrin-1 be an underlying mechanism of secondary injury in the VPN post stroke.
脑梗死后的神经功能缺损不仅与原发性损伤有关,还与梗死灶相关的远隔部位的继发性神经元凋亡有关。轴突导向所必需的 netrin-1 通过与其受体Deleted in Colorectal Cancer(DCC)和Uncoordinated gene 5H(UNC5H)相互作用。当未与 netrin-1 结合时,DCC 和 UNC5H 也是诱导细胞凋亡的依赖性受体。本研究旨在探讨 netrin-1 及其受体在高血压大鼠脑梗死后同侧腹后丘脑核(VPN)损伤中的作用。血管性高血压 Sprague-Dawley 大鼠接受大脑中动脉闭塞(MCAO)。MCAO 后 24 小时给予 netrin-1(600ng/d,持续 7 天)或载体(IgG/Fc)的连续脑室内输注。MCAO 后 8 天和 14 天通过姿势反射评估神经功能。然后,用免疫组化法测定同侧 VPN 中的 NeuN、胶质纤维酸性蛋白、netrin-1 及其受体(DCC 和 UNC5H2),用末端脱氧核苷酸转移酶介导的地高辛-dUTP-生物素缺口末端标记(TUNEL)法检测凋亡,并用 Western blot 分析定量检测 caspase-3、netrin-1、DCC 和 UNC5H2 的表达。MCAO 导致 8 天和 14 天后姿势反射受损,同侧 VPN 中标记神经元的 NeuN 减少,TUNEL 阳性细胞增多,caspase-3 和 UNC5H2 蛋白水平升高,但 DCC 或 netrin-1 表达无明显变化。外源性 netrin-1 输注可增加同侧 VPN 中的神经元数量,减少 TUNEL 阳性细胞数和 caspase-3 蛋白水平,而 UNC5H2 表达不受影响,同时改善姿势反射受损。综上所述,本研究表明,外源性 netrin-1 可能通过其受体 UNC5H2 减轻局灶性脑梗死后同侧 VPN 中的继发性细胞凋亡,从而挽救神经元丢失,提示相对不足的内源性 netrin-1 是 VPN 脑梗死后继发性损伤的潜在机制。