LoPachin R M, Gaughan C L, Lehning E J, Weber M L, Taylor C P
Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY 10467, USA.
Neuroscience. 2001;103(4):971-83. doi: 10.1016/s0306-4522(01)00035-5.
The pathophysiology of brain ischemia and reperfusion injury involves perturbation of intraneuronal ion homeostasis. To identify relevant routes of ion flux, rat hippocampal slices were perfused with selective voltage- or ligand-gated ion channel blockers during experimental oxygen-glucose deprivation and subsequent reperfusion. Electron probe X-ray microanalysis was used to quantitate water content and concentrations of Na, K, Ca and other elements in morphological compartments (cytoplasm, mitochondria and nuclei) of individual CA1 pyramidal cell bodies. Blockade of voltage-gated channel-mediated Na+ entry with tetrodotoxin (1 microM) or lidocaine (200 microM) significantly reduced excess intraneuronal Na and Ca accumulation in all compartments and decreased respective K loss. Voltage-gated Ca2+ channel blockade with the L-type antagonist nitrendipine (10 microM) decreased Ca entry and modestly preserved CA1 cell elemental composition and water content. However, a lower concentration of nitrendipine (1 microM) and the N-, P-subtype Ca2+ channel blocker omega-conotoxin MVIIC (3 microM) were ineffective. Glutamate receptor blockade with the N-methyl-D-aspartate (NMDA) receptor-subtype antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP; 100 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA) receptor subtype blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM/100 microM glycine) completely prevented Na and Ca accumulation and partially preserved intraneuronal K concentrations. Finally, the increase in neuronal water content normally associated with oxygen-glucose deprivation/reperfusion was prevented by Na+ channel or glutamate receptor blockade. Results of the present study demonstrate that antagonism of either postsynaptic NMDA or AMPA glutaminergic receptor subtypes provided nearly complete protection against ion and water deregulation in nerve cells subjected to experimental ischemia followed by reperfusion. This suggests activation of ionophoric glutaminergic receptors is involved in loss of neuronal osmoregulation and ion homeostasis. Na+ channel blockade also effectively diminished neuronal ion and water derangement during oxygen-glucose deprivation and reperfusion. Prevention of elevated Nai+ levels is likely to provide neuroprotection by decreasing presynaptic glutamate release and by improving cellular osmoregulation, adenosine triphosphate utilization and Ca2+ clearance. Thus, we suggest that voltage-gated tetrodotoxin-sensitive Na+ channels and glutamate-gated ionotropic NMDA or AMPA receptors are important routes of ion flux during nerve cell injury induced by oxygen-glucose deprivation/reperfusion.
脑缺血再灌注损伤的病理生理学涉及神经元内离子稳态的紊乱。为了确定相关的离子通量途径，在实验性氧糖剥夺及随后的再灌注过程中，用选择性电压门控或配体门控离子通道阻滞剂灌注大鼠海马切片。采用电子探针X射线微分析法对单个CA1锥体细胞体的形态学区域（细胞质、线粒体和细胞核）中的水分含量以及钠、钾、钙和其他元素的浓度进行定量分析。用河豚毒素（1微摩尔）或利多卡因（200微摩尔）阻断电压门控通道介导的钠离子内流，可显著减少所有区域神经元内过量的钠和钙蓄积，并减少相应的钾流失。用L型拮抗剂尼群地平（10微摩尔）阻断电压门控钙通道可减少钙内流，并适度维持CA1细胞的元素组成和水分含量。然而，较低浓度的尼群地平（1微摩尔）和N-、P-亚型钙通道阻滞剂ω-芋螺毒素MVIIC（3微摩尔）无效。用N-甲基-D-天冬氨酸（NMDA）受体亚型拮抗剂3-（2-羧基哌嗪-4-基）丙基-1-膦酸（CPP；100微摩尔）或α-氨基-3-羟基-5-甲基-4-异唑丙酸（AMPA）受体亚型阻滞剂6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX；10微摩尔/100微摩尔甘氨酸）阻断谷氨酸受体，可完全阻止钠和钙的蓄积，并部分维持神经元内钾浓度。最后，钠通道或谷氨酸受体阻断可防止通常与氧糖剥夺/再灌注相关的神经元水分含量增加。本研究结果表明，拮抗突触后NMDA或AMPA谷氨酸能受体亚型可几乎完全保护遭受实验性缺血再灌注的神经细胞免受离子和水分失调的影响。这表明离子型谷氨酸能受体的激活与神经元渗透调节和离子稳态的丧失有关。钠通道阻断在氧糖剥夺和再灌注期间也有效地减少了神经元离子和水分紊乱。防止细胞内钠离子水平升高可能通过减少突触前谷氨酸释放以及改善细胞渗透调节、三磷酸腺苷利用和钙清除来提供神经保护。因此，我们认为电压门控的对河豚毒素敏感的钠通道以及谷氨酸门控的离子型NMDA或AMPA受体是氧糖剥夺/再灌注诱导的神经细胞损伤期间重要的离子通量途径。
