镁离子降低耳蜗谷氨酸兴奋毒性及其对耳蜗神经元细胞钙离子内流的影响

Effect of magnesium sulfute on glutamate-induced cochlear neurotoxicity and intracellular Ca^(2+) in spiral ganglion neuron

目的:观察硫酸镁对培养耳蜗神经细胞谷氨酸兴奋毒性的保护作用。方法:将体外培养新生大鼠耳蜗Corti器分为加入0.1mmol·L-1谷氨酸组和不同浓度(0.5、1.0、2.0和4.0mmol·L-1)硫酸镁组以及谷氨酸和硫酸镁联合组,采用Neurofilament 200kDa免疫荧光染色标记神经元和神经纤维,观察细胞形态和神经元存活数目;采用Fluo-3/AM染色法和激光共聚焦显微镜测定谷氨酸和硫酸镁作用组神经细胞内游离钙的变化。结果:谷氨酸作用24h后,神经元胞体变得不规则,数目明显减少,神经末梢出现肿胀。0.1mmol·L-1谷氨酸联合0.5、1.0和2.0mmol·L-1硫酸镁组的耳蜗神经元存活数量与单纯谷氨酸组比较明显增多(P<0.01)。单独硫酸镁作用耳蜗,2.0mmol·L-1浓度以下未见明显毒性。谷氨酸作用前应用硫酸镁在0.5~4h时间段观察到神经元钙离子内流低于单纯谷氨酸作用组(P<0.01)。单独应用硫酸镁对细胞内钙无影响。结论:硫酸镁对培养耳蜗神经细胞谷氨酸兴奋毒性具有显著的保护作用,其机制可能与镁离子抑制细胞内钙累积有关。

Objective To investigate the protective effects of magnesium sulfate on glutamate-induced neurotoxicity in cultured cochlear. Methods The cortis of newborn rats were cultivated in vitro. The cultured cochlear neurons were divided into glutamate group treated with 0.1 mmol·L-1 glutamate, magnesium sulfate group treated with different concentrations of magnesium sulfate （0.5, 1.0, 2.0 and 4.0 mmol·L-1 ） and combined group treated with glutamate and magnesium sulfate for 24 h. immunofluorescence staining of Neurofilament 200kDa antibody was used to label spiral ganglion neuron （SGN） and the terminate of dendrites. The morphological changes of SGN were examined by fluorescent microscope and the amount of SGN was determined. In addition, the SGN was loaded with 10 mmol·L-1 Fluo-3 /AM, and the intracellular Ca^2＋ was measured by laser confocal scanning microscope （LCSM）. Results Many SGN were smaller and swelling and vacuoles at the terminate of dendrites following glutamate treatment for 24 h. In combined group treated with glutamate and 0.5, 1.0 and 2.0 mmol·L-1 of magnesium sulfate, the amounts of SGN increased compared with glutamate group （P 〈 0.01 ）. Under the concentration of 2 mmol·L-1, magnesium sulfate was safe to SGN survival. Magnesium sulfate reduced the calcium influx of SGN from 0.5 to 4 h following glutamate treatment. Compared with glutamate group, thesignificant decrease of cell fluorescence intensity of SGN was observed in Conclusion Magnesium sulfate provides protective effects against SGN damage by It can inhibit intracellular Ca^2＋ accumulation. combined group （P 〈 0.01）. glutamate-induced neurotoxicity.