BRAIN RESEARCH ELSEVIER Brain Research 663 (1994) 329-334 Short communication Anoxia induces an increase in intracellular sodium in rat central neurons in vitro Jonathan E. Friedman *, Gabriel G. Haddad Department of Pediatrics, Section of Respiratory Medicine and Department of Cellular and Molecular Physiology, Yale University School of Medicine, Fitkin Bldg. 517, 333 Cedar Str., New Haven, CT06520, USA Accepted 16 August 1994 Abstract Following our previous observations that anoxia induces a drop in extracellular Na ÷ in the brain slice and that removal of extracelluar Na + prevents the anoxia-induced morphological changes in dissociated hippocampal neurons, we hypothesized that intraceilular Na ÷ increases during anoxia in isolated neurons. Using the fluorophore Sodium Green in freshly dissociated rat CA1 neurons, and SBFI in cultured cortical neurons, we found that 10 min of anoxia caused an increase in Na~- in both types of cells, with a latency of about 2 min. In CA1 neurons, fluorescence increased by an average of 20.34% (n = 8). The mean baseline Na~- level (determined using SBFI) was 25 + 2.5 raM, which increased to about an average of 52 + 3 mM after 3-4 rain. These and our previous results strongly suggest that Na+-mediated events are involved in anoxia-induced nerve injury. Keywords: Sodium Green; Cortex; Hippocampus; Volume Oxygen deprivation has long been known to cause irreversible damage to the mammalian brain. This damage is believed to result primarily from a massive influx of calcium into nerve cells, mediated in part by the excitatory amino acid glutamate [6]. Calcium, by virtue of its ability to activate, for example, enzymes such as lipases, proteases and kinases has thus been considered to cause cell damage. However, we, as well as others, have shown in vitro that nerve damage from anoxia still occurs in the absence of extraceliular Ca 2+, and in the absence of any detectable rise in intra- cellular Ca 2÷ [10,11]. Since (a) we have previously shown that extracellular Na ÷ decreases in the brain slice during anoxia [19], and (b) our previous data indicate that extracellular Na ÷ is important in the anoxia-induced depolarization [16], we entertained the hypothesis that Na + enters into neurons during 0 2 deprivation. In order to address this hypothesis, we have carried out studies primarily using freshly dissociated (i.e. iso- lated) CA1 hippocampal pyramidal neurons. This is an optimal system for testing the direct effect of anoxia on * Corresponding author. Fax: (1) (203) 785-6337. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01000-5 cells, since potentially confounding factors such as synaptic transmission and release of excitatory amino acids are obviated. In addition, CA1 pyramidal neu- rons are considered to be among the most sensitive cells to anoxia [23,31]. Using this system, and the Na÷-sensitive fluorophore Sodium Green (NaGrn), we have monitored changes in Na~-. However, since Na- Grn is not a ratiometric dye, we also employed another Na+-sensitive dye, SBFI (sodium-binding benzofuran isophthalate), in order to obtain quantitative results [17,27]. Dissociated cell preparation and loading with Sodium green. Cells were prepared as previously reported [11]. In brief, 21-30 day old Sprague-Dawley rats were anesthetized by inhalation of methoxyfluorane (Pit- mann-Moore), decapitated, the brain rapidly removed and placed into ice-cold oxygenated dissociation buffer (DB) (120 mM NaC1, 5 mM KC1, 1 mM CaC12, 1 mM MgC12, 25 mM glucose and 20 mM HEPES (Sigma), pH 7.4). The hippocampi were removed and cut into 400-500 /.~m slices, which were immediately placed into a dissociation chamber containing 10 ml of DB + 10 mg trypsin (Sigma) for 40 min followed by the addition of 4 mg Pronase E (Sigma) for another 20 min. The buffer was replaced with fresh DB, without