Department of Ophthalmology, Osaka Medical College, Takatsuki, Osaka, Japan.
Invest Ophthalmol Vis Sci. 2010 Jun;51(6):3236-43. doi: 10.1167/iovs.09-4192. Epub 2010 Jan 13.
To investigate the hypoxia-induced death of rat retinal neurons and to determine whether P2X(7) activation is involved in this type of neuronal death.
Cultured retinal neurons from fetal rats were used. The effects and time course of various degrees of hypoxia (1%-5% O(2)) in the death of retinal neurons, were examined. The effects of P2X(7) antagonists, oxidized adenosine triphosphate (oxidized ATP; 30-100 microM), and brilliant blue G (BBG; 100 nM-10 microM) on hypoxia-induced neuronal death, including apoptosis, were assessed by using trypan blue exclusion, TUNEL assays, and cleaved caspase-3 immunoreactivity. Immunocytochemical analysis was performed to determine whether these neurons express P2X(7) receptors. The effects of P2X(7) receptor stimulation, induced by the P2X(7) agonist benzoyl- benzoyl-ATP (BzATP), on neuronal viability and intracellular Ca(2+) levels (Ca(2+)) were examined.
Retinal neuronal death increased according to the degree of hypoxia and became more severe after 12 hours. Both oxidized ATP and BBG significantly decreased hypoxia-induced neuronal death. Immunocytochemistry demonstrated that P2X(7) receptors were expressed by the cultured retinal neurons. ATP and BzATP caused P2X(7) receptor-dependent neuronal death in a dose-dependent manner and led to a sustained increase in Ca(2+), with BzATP being more effective than ATP. These effects were hypoxia-induced factor-1alpha- independent and were prevented by oxidized ATP.
The results suggest that the death of retinal neurons can be triggered by hypoxia and that P2X(7) activation is involved in the hypoxia-induced death of retinal neurons. P2X(7) antagonists can prevent hypoxia-induced damage in retinal neurons.
研究缺氧诱导的大鼠视网膜神经元死亡，并确定 P2X(7) 激活是否参与这种类型的神经元死亡。
使用来自胎鼠的培养视网膜神经元。检查不同程度缺氧（1%-5% O(2)) 对视网膜神经元死亡的影响和时间进程。通过台盼蓝排斥试验、TUNEL 测定和 cleaved caspase-3 免疫反应性评估 P2X(7) 拮抗剂、氧化三磷酸腺苷（氧化 ATP；30-100 microM) 和亮蓝 G (BBG；100 nM-10 microM) 对缺氧诱导的神经元死亡（包括细胞凋亡）的影响。进行免疫细胞化学分析以确定这些神经元是否表达 P2X(7) 受体。通过 P2X(7) 激动剂苯甲酰-苯甲酰三磷酸（BzATP）刺激 P2X(7) 受体，观察其对神经元活力和细胞内 Ca(2+) 水平 (Ca(2+)) 的影响。
视网膜神经元死亡随缺氧程度增加而增加，12 小时后变得更加严重。氧化 ATP 和 BBG 均显著降低缺氧诱导的神经元死亡。免疫细胞化学显示培养的视网膜神经元表达 P2X(7) 受体。ATP 和 BzATP 以剂量依赖的方式引起 P2X(7) 受体依赖性神经元死亡，并导致 Ca(2+) 持续增加，BzATP 比 ATP 更有效。这些作用与缺氧诱导因子-1alpha 无关，可被氧化 ATP 阻止。
结果表明，视网膜神经元的死亡可被缺氧触发，P2X(7) 激活参与缺氧诱导的视网膜神经元死亡。P2X(7) 拮抗剂可预防缺氧诱导的视网膜神经元损伤。
