Hewett S J, Muir J K, Lobner D, Symons A, Choi D W
Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA.
Stroke. 1996 Sep;27(9):1586-91. doi: 10.1161/01.str.27.9.1586.
Previous studies have shown that brain ischemia and other insults can induce a marked increase in inducible nitric oxide synthase (iNOS) expression in astrocytes and some immune cells, but the biological significance of this phenomenon has not been elucidated. The purpose of the present study was to determine whether this induction of astrocyte iNOS alters neuronal vulnerability to severe hypoxic insults.
Astrocytic iNOS was induced by exposure of murine cortical cultures to interferon gamma in combination with either interleukin-1 beta or lipopolysaccharide. Cultures were exposed to combined oxygen-glucose deprivation. The extracellular concentration of glutamate was measured by high-performance liquid chromatography. N-Methyl-D-aspartate (NMDA) receptor activity was assessed by measurement of 45Ca2+ influx: neuronal death was assessed by morphological examination and quantitated by measurement of lactate dehydrogenase efflux to the bathing medium.
In murine neocortical cell cultures containing neurons and astrocytes, neuronal injury induced by combined oxygen-glucose deprivation was not reduced by the addition of the nitric oxide synthase inhibitors NG-nitro-L-arginine or LG-nitro-arginine methyl ester. However, after induction of astrocyte iNOS activity with interferon gamma plus lipopolysaccharide or interleukin-1 beta, oxygen-glucose deprivation-induced neuronal injury was markedly enhanced and nitric oxide synthase inhibitors became protective. This iNOS-mediated potentiation was associated with a large increase in both extracellular glutamate accumulation and 45Ca2+ influx into neurons. The potentiation could be blocked by MK-801 but not CNQX, suggesting critical involvement of NMDA receptor activation.
These results support the idea that nitric oxide production mediated by induced astrocytic iNOS can potentiate NMDA receptor-mediated neuronal death consequent to hypoxic-ischemic insults.
以往研究表明，脑缺血及其他损伤可诱导星形胶质细胞和一些免疫细胞中诱导型一氧化氮合酶（iNOS）表达显著增加，但该现象的生物学意义尚未阐明。本研究旨在确定星形胶质细胞iNOS的这种诱导是否会改变神经元对严重缺氧损伤的易感性。
通过将小鼠皮质培养物暴露于γ干扰素联合白细胞介素-1β或脂多糖来诱导星形胶质细胞iNOS。培养物暴露于联合氧-葡萄糖剥夺环境。通过高效液相色谱法测量细胞外谷氨酸浓度。通过测量45Ca2+内流评估N-甲基-D-天冬氨酸（NMDA）受体活性：通过形态学检查评估神经元死亡，并通过测量乳酸脱氢酶向培养基中的外渗进行定量。
在含有神经元和星形胶质细胞的小鼠新皮质细胞培养物中，联合氧-葡萄糖剥夺诱导的神经元损伤并未因添加一氧化氮合酶抑制剂NG-硝基-L-精氨酸或LG-硝基-精氨酸甲酯而减轻。然而，在用γ干扰素加脂多糖或白细胞介素-1β诱导星形胶质细胞iNOS活性后，氧-葡萄糖剥夺诱导的神经元损伤明显增强，一氧化氮合酶抑制剂变得具有保护作用。这种iNOS介导的增强作用与细胞外谷氨酸积累和45Ca2+流入神经元的大量增加有关。这种增强作用可被MK-801阻断，但不能被CNQX阻断，提示NMDA受体激活起关键作用。
这些结果支持以下观点，即诱导的星形胶质细胞iNOS介导的一氧化氮生成可增强缺氧缺血性损伤后NMDA受体介导的神经元死亡。
