Chen Yi-Ning, Yamada Hideyuki, Mao Wei, Matsuyama Shigemi, Aihara Makoto, Araie Makoto
Department of Ophthalmology, University of Tokyo School of Medicine, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan.
Brain Res. 2007 May 7;1148:28-37. doi: 10.1016/j.brainres.2007.02.027. Epub 2007 Feb 22.
Hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. However, the precise mechanism of death signaling and how neuroprotective agents affect it are still unclear. The aim of this study is to characterize the mechanisms of hypoxia-induced apoptosis of cultured purified RGCs and to study the neuroprotective effects of beta-adrenergic antagonists. Rat RGCs were purified utilizing a modified two-step immuno-panning procedure. First, the extent of apoptosis in RGCs under hypoxia was quantified. Next, the effects of glutamate-channel antagonists (MK801 or DNQX), Bax inhibiting peptide (BIP), and beta-adrenergic antagonists (betaxolol, nipradilol, timolol or carteolol) on hypoxia-induced RGC death were investigated by the cell viability assay. Third, the effects of beta-adrenergic antagonists on hypoxia-induced increase of intracellular calcium concentrations (Ca(2+)) and the additional effect of NO scavenger to nipradilol were evaluated. Apoptotic RGC percentages under hypoxia were significantly increased compared to the control. The viability of RGCs under hypoxia was not affected by MK801 or DNQX, whereas it was increased in a dose-dependent manner with exposure to BIP, and to betaxolol, nipradilol, timolol, but not to carteolol. These effective beta-adrenergic antagonists showed no significant change in hypoxia-induced Ca(2+) levels. The NO scavenger alleviated neuroprotective effect by nipradilol. In conclusion, purified RGC damage induced by hypoxia involves Bax-dependent apoptotic pathway, but mostly independent of glutamate receptor-mediated excitotoxicity. Betaxolol, timolol and nipradilol showed a protective effect against hypoxia-induced RGC death, which was thought to be irrelevant either to calcium channel or beta-adrenoceptor blocking effects.
缺氧诱导的视网膜神经节细胞（RGC）死亡与青光眼性视神经病变有关。然而，死亡信号传导的确切机制以及神经保护剂如何影响该机制仍不清楚。本研究的目的是阐明培养的纯化RGC缺氧诱导凋亡的机制，并研究β-肾上腺素能拮抗剂的神经保护作用。利用改良的两步免疫淘选法纯化大鼠RGC。首先，对缺氧条件下RGC中的凋亡程度进行定量。其次，通过细胞活力测定法研究谷氨酸通道拮抗剂（MK801或DNQX）、Bax抑制肽（BIP）和β-肾上腺素能拮抗剂（倍他洛尔、尼普地洛、噻吗洛尔或卡替洛尔）对缺氧诱导的RGC死亡的影响。第三，评估β-肾上腺素能拮抗剂对缺氧诱导的细胞内钙浓度（Ca(2+)）升高的影响以及NO清除剂对尼普地洛的附加作用。与对照组相比，缺氧条件下凋亡RGC的百分比显著增加。缺氧条件下RGC的活力不受MK801或DNQX的影响，而暴露于BIP、倍他洛尔、尼普地洛、噻吗洛尔时，其活力呈剂量依赖性增加，但卡替洛尔无此作用。这些有效的β-肾上腺素能拮抗剂在缺氧诱导的Ca(2+)水平上无显著变化。NO清除剂减轻了尼普地洛的神经保护作用。总之，缺氧诱导的纯化RGC损伤涉及Bax依赖的凋亡途径，但大多独立于谷氨酸受体介导的兴奋性毒性。倍他洛尔、噻吗洛尔和尼普地洛对缺氧诱导的RGC死亡具有保护作用，这被认为与钙通道或β-肾上腺素能受体阻断作用无关。
