Aas J E, Berg-Johnsen J, Hegstad E, Laake J H, Langmoen I A, Ottersen O P
Department of Anatomy, University of Oslo, Norway.
J Cereb Blood Flow Metab. 1993 May;13(3):503-15. doi: 10.1038/jcbfm.1993.65.
This study was undertaken to elucidate the roles of neurons and glial cells in the handling of glutamate and glutamine, a glutamate precursor, during cerebral ischemia. Slices (400-600 microns) from human neocortex obtained during surgery for epilepsy or brain tumors were incubated in artificial cerebrospinal fluid and subjected to 30 min of combined hypoxia and glucose deprivation (an in vitro model of brain ischemia). These slices, and control slices that had not been subjected to "ischemic" conditions, were then fixed and embedded. Ultrathin sections were processed according to a postembedding immunocytochemical method with polyclonal antibodies raised against glutamate or glutamine, followed by colloidal gold-labeled secondary antibodies. The gold particle densities over various tissue profiles were calculated from electron micrographs using a specially designed computer program. Combined hypoxia and glucose deprivation caused a reduced glutamate immunolabeling in neuronal somata, while that of glial processes increased. Following 1 h of recovery, the glutamate labeling of neuronal somata declined further to very low values, compared to control slices. The glutamate labeling of glial cells returned to normal levels following recovery. In axon terminals, no consistent change in the level of glutamate immunolabeling was observed. Immunolabeling of glutamine was low in both nerve terminals and neuronal somata in normal slices and was reduced to nondetectable levels in nerve terminals upon hypoxia and glucose deprivation. This treatment was also associated with a reduced glutamine immunolabeling in glial cells. Reversed glutamate uptake due to perturbations of the transmembrane ion concentrations and membrane potential probably contributes to the loss of neuronal glutamate under "ischemic" conditions. The increased glutamate labeling of glial cells under the same conditions can best be explained by assuming that glial cells resist a reversal of glutamate uptake, and that their ability to convert glutamate into glutamine is compromised due to the energy failure. The persistence of a nerve terminal pool of glutamate is compatible with recent biochemical data indicating that the exocytotic glutamate release is contingent on an adequate energy supply and therefore impeded during ischemia.
本研究旨在阐明在脑缺血期间神经元和神经胶质细胞在处理谷氨酸及其前体谷氨酰胺中的作用。从因癫痫或脑肿瘤手术获取的人类新皮质切取薄片（400 - 600微米），置于人工脑脊液中，并进行30分钟的缺氧和葡萄糖剥夺处理（一种脑缺血的体外模型）。然后将这些薄片以及未经历“缺血”条件的对照薄片进行固定和包埋。超薄切片按照包埋后免疫细胞化学方法处理，使用针对谷氨酸或谷氨酰胺的多克隆抗体，随后用胶体金标记的二抗。利用专门设计的计算机程序从电子显微照片计算各种组织轮廓上的金颗粒密度。缺氧和葡萄糖剥夺联合处理导致神经元胞体中谷氨酸免疫标记减少，而神经胶质细胞突起的免疫标记增加。恢复1小时后，与对照薄片相比，神经元胞体的谷氨酸标记进一步下降至非常低的值。恢复后神经胶质细胞的谷氨酸标记恢复到正常水平。在轴突终末，未观察到谷氨酸免疫标记水平有一致变化。正常薄片中神经终末和神经元胞体的谷氨酰胺免疫标记均较低，缺氧和葡萄糖剥夺后神经终末的谷氨酰胺免疫标记降至不可检测水平。这种处理还与神经胶质细胞中谷氨酰胺免疫标记减少有关。跨膜离子浓度和膜电位紊乱导致的谷氨酸摄取逆转可能是“缺血”条件下神经元谷氨酸丢失的原因。在相同条件下神经胶质细胞谷氨酸标记增加，最合理的解释是假设神经胶质细胞抵抗谷氨酸摄取逆转，并且由于能量衰竭其将谷氨酸转化为谷氨酰胺的能力受损。谷氨酸神经终末池的持续存在与最近的生化数据一致，这些数据表明谷氨酸的胞吐释放取决于充足的能量供应，因此在缺血期间受到阻碍。
