Friedman J E, Haddad G G
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510.
J Neurosci. 1993 Jan;13(1):63-72. doi: 10.1523/JNEUROSCI.13-01-00063.1993.
Although we and others have previously shown that newborn central mammalian neurons are more tolerant to anoxia than their adult counterparts, we do not know whether neonatal nerve cells accumulate free cytosolic calcium (Ca2+i) less than adults in response to O2 deprivation. In order to determine whether anoxia increases Ca2+i in adult and neonatal neurons, we monitored calcium in CA1 hippocampal neurons using the calcium-sensitive probe fluo-3 and confocal microscopy. These neurons were studied in the dissociated state in order to study their inherent response to anoxia without the influence of modulatory factors such as synaptic input and neurotransmitters. Severe anoxia caused a rapid increase in Ca2+i in adult CA1 hippocampal neurons, followed by swelling and bleb formation. In neonatal neurons, the latency of this calcium rise was about five times longer than in the adult. Removal of extracellular calcium and addition of calcium channel blockers (Co2+) greatly attenuated the increase in Ca2+i in response to anoxia but did not prevent cell swelling and injury. The addition of glutamate antagonists MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione did not affect the increase in Ca2+i induced by anoxia. Replacing extracellular sodium with impermeant cations (N-methyl-D-glucamine) prevented anoxia-induced nerve injury. In addition, Ca2+i levels dropped, rather than increased, during the anoxic period in the absence of sodium; Ca2+i returned toward baseline levels upon reoxygenation. Glutamate was also studied, in the presence of O2, to compare with the effects of anoxia. Adult neurons responded with similar latencies to both anoxia and glutamate (10-500 microM). Neonatal neurons, however, exhibited a more rapid increase in Ca2+i in response to glutamate than they did when exposed to anoxia. We conclude (1) that neonatal neurons increase Ca2+i more slowly than adults in response to anoxia and that this response is due to an influx of Ca2+o into the cytosol, (2) that an increase in Ca2+i is not necessary to induce nerve injury when nerve cells are deprived of O2, (3) that the removal of Nao+ prevents cell swelling and blebbing and is associated with a decrease in Ca2+i during anoxia, and finally (4) that glutamate exposure may not be a completely valid model for the direct action of anoxia on neurons.
尽管我们和其他人之前已经表明，新生哺乳动物中枢神经元比成年神经元对缺氧更具耐受性，但我们不知道新生儿神经细胞在缺氧时胞质游离钙（Ca2+i）的积累是否比成年人少。为了确定缺氧是否会增加成年和新生神经元中的Ca2+i，我们使用钙敏探针Fluo-3和共聚焦显微镜监测海马CA1神经元中的钙。这些神经元在解离状态下进行研究，以便研究它们对缺氧的内在反应，而不受突触输入和神经递质等调节因素的影响。严重缺氧导致成年海马CA1神经元中的Ca2+i迅速增加，随后出现肿胀和气泡形成。在新生神经元中，这种钙升高的潜伏期比成年神经元长约五倍。去除细胞外钙并添加钙通道阻滞剂（Co2+）可大大减弱缺氧引起的Ca2+i增加，但不能防止细胞肿胀和损伤。添加谷氨酸拮抗剂MK-801和6-氰基-7-硝基喹喔啉-2,3-二酮不影响缺氧诱导的Ca2+i增加。用不透性阳离子（N-甲基-D-葡萄糖胺）替代细胞外钠可防止缺氧诱导的神经损伤。此外，在无钠的缺氧期间，Ca2+i水平下降而非升高；复氧后Ca2+i恢复到基线水平。还研究了在有氧存在的情况下谷氨酸，以与缺氧的影响进行比较。成年神经元对缺氧和谷氨酸（10-500 microM）的反应潜伏期相似。然而，新生神经元对谷氨酸的反应比暴露于缺氧时Ca2+i增加更快。我们得出结论：（1）新生神经元对缺氧的反应中Ca2+i增加比成年神经元慢，且这种反应是由于Ca2+o流入细胞质所致；（2）当神经细胞缺氧时，Ca2+i增加并非诱导神经损伤所必需；（3）去除Nao+可防止细胞肿胀和气泡形成，并与缺氧期间Ca2+i减少有关；最后（4）谷氨酸暴露可能不是缺氧对神经元直接作用的完全有效模型。
