Involvement of platelet-activating factor (PAF) in glutamate neurotoxicity in neuronal sultures

为了阐明血小板活化因子（PAF）在谷氨酸神经毒性中的作用，采用原代神经元培养进行了体外实验。在胚胎大鼠大脑皮层分离的原代神经元培养物中，检测了抗PAF免疫球蛋白g （aPAF-IgG）和三种PAF受体拮抗剂（BN52021、CV6209和E5880）的神经保护活性。将培养的神经元置于谷氨酸（1mM）中60min。暴露24小时后，aPAF-1gG以剂量依赖的方式显示出对神经元损伤的保护作用。三种PAF拮抗剂对培养物也有保护作用（1杯/ml aPAF处理P<0.05, 100 muM BN52021处理P<0.01, 10 nM CV6209处理P<0.05, 10 nM E5880处理P<0.01）。Fura-2检测用于评估低剂量外源性PAF是否影响培养的神经元。培养的神经元被加载Fura-2/AM。预孵育120分钟后，负载fura -2的神经元暴露于不同浓度的PAF中60分钟。通过测量代表Fura-2从受损神经元释放量的培养基的荧光强度，我们检测到即使在低剂量的PAF下Fura-2的释放也增加（在10 nM PAF下P<0.01）。我们进一步研究了神经元对谷氨酸的反应产生PAF。谷氨酸暴露培养基中PAF水平显著高于未暴露培养基（P<0.05）。我们的研究结果表明PAF在谷氨酸神经毒性中起重要作用。

To clarify the role platelet-activating factor (PAF) in glutamate neurotoxicity, in vitro experiments using primary neuronal cultures were performed. The anti-PAF immunoglobulin-G (aPAF-IgG) and the three PAF receptor antagonists (BN52021, CV6209, and E5880) were tested for their neuroprotective activity in primary neuronal cultures isolated from embryonic rat cerebral cortex. The cultured neurons were exposed to glutamate (1mM) for 60min. Twenty-four hours after this exposure, aPAF-1gG demonstrated evidence of protective effects against neuronal damage in a dose-dependent manner. Protective effects aiso were observed in cultures treated with the three PAF antagonists (P<0.05 at 1 mug/ml aPAF P<0.01 at 100 muM BN52021, P<0.05 at 10 nM CV6209 and P<0.01 at 10 nM E5880). The Fura-2 assay was used to estimate whether low dosages of exogenous PAF affect cultured neurons. The cultured neurons were loaded with Fura-2/AM.After preincubation for 120 min, the Fura-2-loaded neurons were exposed to various concentrations of PAF for 60 min. By measuring the fluorescent intensity of the medium as representing the amount of Fura-2-released from damaged neurons, we detected an increased release of Fura-2, even at low doses of PAF (P<0.01 at 10 nM PAF). We further studied PAF production by neurons in response to glutamate. The level of PAF measured in the medium exposed to glutamate was significantly higher than the level in the medium unexposed to glutamate (P<0.05). Our results suggest an important role of PAF in glutamate neurotoxicity.