A.C.H. Yu. L. Hertz, M.D. Norenberg. E. Sykova and S.G. Waxman (Eds.) Progress in Bruin Research. Vol. 94 0 1992 Elsevier Science Publishers B.V. All rights reserved.
69
CHAPTER 6
Swelling of C6 glioma cells and astrocytes from glutamate, high K + concentrations or acidosis 0. Kernpskil, F. Staub, G.-H. Schneider, H. Weigt and A. Baethmann
’
Institute f o r Surgical Research, Ludwig-Maximilians University, Klinikum Grosshadern, 0-8000Miinchen 70, and Institute of Neurosurgical Pathophysiology, Johannes Gutenberg-University, 0-6500Mainz, Germany
Introduction
During pathophysiologic events such as ischemia the control of glial cell volume is lost. The swelling of cellular elements in the brain - glial endfeet but also dendrites - is referred to as “cytotoxic brain edema”. Brain edema and the resulting rise in intracranial pressure (ICP) often determine the ultimate outcome of neurosurgical patients. So far, it has been the general understanding that with impaired energy supply, cell swelling will result from the failure of Na+/K+-ATPase according to the pump-leak model of cell volume regulation (Macknight and Leaf, 1977). Glial swelling in cerebral ischemia, however, occurs fast, within 5 min after interruption of the energy supply. In vitro, on the other hand, the onset of swelling depends on the size of the volume of the incubation medium, as elegantly demonstrated by Ames and Nesbett (1983a - c) using the isolated retina. Their observations are best explained by assuming that toxic compounds accumulating in the extracellular environment mediate cell swelling and also nerve cell death. Ames’ data are in line with a variety of other observations which have led to the concept of mediators of secondary brain damage (Baethmann et al., 1980). Mediators are substances released or activated during pathophysiologic events such as ischemia. Excitotoxins (e.g., glutamate), lactic acidosis, free fatty acids, and elevated extracellular
(ex.) concentrations of potassium are the most prominent mediator candidates identified so far. Reports on the occurrence of these mediators under ischemic conditions in vivo are available in abundance. Unfortunately, it is virtually impossible in vivo to discriminate the effects of single mediators on glial volume, since, in addition to multiple interactions, they are released or activated practically simultaneously. In a series of in vitro studies, we have, therefore, analyzed glial cell volume changes during defined and strictly controlled alterations of the extracellular environment in vitro (Kempski et al., 1983, 1987, 1988a,b, 1991; Staub et al., 1990). This article is an attempt to summarize the results. Methods
Glial cell culture Experiments were performed with C6 glioma cells (Benda et al., 1968) and astrocytes from primary culture. The latter were obtained from 3-day-old neonatal rats employing a method adapted from Frangakis and Kimelberg (Frangakis and Kimelberg, 1984; Staub et al., 1990). In brief, forebrains were freed from meninges and choroid plexus under a dissectingmicroscope. The tissue was chopped to fine pieces, and repeatedly digested using dispase. Suspended cells were sedimented and seeded into Petri dishes. The purity of all prepara-
70
tions was assessed by GFAP PAP stains (DAKO). Both cell types were cultured in monolayers in Petri dishes using Dulbecco’s minimal essential medium (DMEM), supplemented with penicillin G (100 IU/ml) and streptomycin (50 pg/ml). Cell growth and differentiation was stimulated by 10% fetal calf serum (FCS). Cells were cultured at 37°C in a humidified atmosphere of 5% CO, and 95% room air. The culture medium was changed and C6 cells were subcultivated daily.
Measurement of cell volume Cell volume was determined by use of a flow cytometer (MetrizellR)combining the Coulter principle with a hydrodynamic focusing technique (Kachel, 1976). The device permits to detect even subtle cell volume changes of 1-2070 with high precision. Latex beads of known diameter served to calibrate the system.
Cell suspension Cells were harvested for the experiments 2 days after subcultivation when the Petri dishes showed confluent monolayers and cells had generated glial processes. This method ensured a fairly constant cell count in the incubation chamber during the various experiments. In primary astrocyte cultures “cell differentiation” was induced 24 h before harvesting by addition of dibutyrylic-cyclic-AMP (dB-CAMP) at a final concentration of 0.5 mM in the medium (Fedoroff et al., 1984). After enzymatic detachment from the Petri dishes with trypsin-EDTA, cells were washed in DMEM + 10% FCS to inactivate trypsin. Then the cells were resuspended, rinsed and centrifuged in FCS-free experimental medium two times.
The experimental mode1 The experimental model permits to quantify cell volume changes at a far higher level of accuracy than possible under pathologic conditions in vivo. Cell volume changes of less than 1% can be detected. Furthermore it is possible to control and maintain important parameters such as ion concentrations, osmolality, pH, PO, and temperature. Suspended C6 cells have a very active oxidative metabolism, as demonstrated by their vivid oxygen consumption (Kempski et al., 1983). The major advantages of using C6 cells are the rapid availability of large cell numbers necessary for reliable flow cytometric volume measurements, and the homogeneous cell size distribution permitting the detection of even subtle volume changes. C6 cells express specific glial markers such as SlOO and GFAP, as well as enzymes, transmitter uptake systems and ion carriers typical for astrocytes (Kempski et al., 1988a). Astrocytes from primary culture are used to verify observations obtained with C6 cells (Kempski et al., 1988a, 1991; Staub et al., 1990; Schneider et al., 1992). So far, astrocytes have always mirrored the observations previously made with C6 cells. It is concluded that the basic mechanisms of cell volume control are very similar for both cell types.
Incubation chamber After the second centrifugation cells were resuspended and quickly introduced into a plexiglass incubation chamber (Kempski et al., 1983). Important extracellular parameters like temperature (37”C), pH and PO, were monitored by electrodes in the chamber thus allowing to keep them constant. A membrane oxygenator from permeable silicon rubber tubing maintains constant partial pressures of 0,, CO, and N,. The medium was buffered with bicarbonate (24 mM), and extracellular pH was kept at 7.4 by regulating the flow of CO, accordingly. A teflon-covered magnetic stirrer in the chamber prevented sedimentation of the suspended cells. Samples of the suspension were retrieved or drugs added through openings in the chamber wall.
Results and discussion
Anoxia The in vitro model was first used to evaluate whether anoxia or inhibition of the cellular energy supply without any further changes of the extracellular environment would be accompanied by swelling (Kempski et al., 1987).Anoxia was induced by discontinuation of the oxygen supply to the mem-
71
brane oxygenator, and subsequent ventilation with nitrogen. In addition the incubation chamber was placed into an air-tight tent flooded with nitrogen to avoid any leakage of oxygen into the chamber. Low oxygen tensions possibly remaining in the medium were consumed by the respiration of the dense cell suspension within minutes. Anoxia was maintained for 2 h - and cell swelling was never observed. The increase of e.c. lactate indicated that anaerobic glycolysis was sufficient to maintain ion gradients and cell volume control. Indeed the analysis of i.c. electrolyte concentrations by atomic absorption spectrometry revealed normal i.c. Na+ concentrations. Therefore, iodoacetate was used to inhibit glycolysis in addition to anoxia. This treatment successfully abolished the i.c./e.c. Na+ gradient within 120 min, but, again, cell volume was unaffected (Kempski et al., 1987). These results are in line with observations of Ames and Nesbett using the isolated retina (1983a - c). In anoxia retinal swelling was absent if the extracellular incubation medium was large enough to dilute toxic material released from the tissue. Medium conditioned by anoxic retinas, or therestriction of the e.c. fluid volume during anoxia was able to enhance tissue swelling. We conclude that in the narrow, restricted e.c. space of the brain in vivo, mediators of secondary brain damage released or activated by pathophysiologic events accumulate to cause glial swelling and, eventually, glial and nerve cell death.
Inhibition of Na+/K+-ATPase by ouabain In another study this conclusion found further support. Here, the sodium-potassium pump was blocked by the cardiac glycoside ouabain. The Na+ gradient was lost by this treatment within 90 min in C6 cells (Kempski et al., 1988b). Intracellular Na+ measurements did demonstrate a near six-fold increase of intracellular Na+ content from a low 13 x mol/cell to 78 x mol/cell, and, hence, proved that the Na+/K+-ATPase was effectively inhibited by 1 mM ouabain. Simultaneously asteep drop in the i.c. potassium content from 190 x mol/cell to 30 x mol/cell was
observed. Forty-five minutes of ouabain treatment were not sufficient to completely collapse the sodium gradient: i.c. Na+ was still 56 x mol/cell. Just as observed with complete anoxia (Kempski et al., 1987), the inhibition of the Na+/K+ pump did not cause cell swelling (Kempski et al., 1988b). Again, the data support the contention that failure of the energy supply per se, or of sodium transport by Na+/K+-ATPase do not suffice to induce glial swelling in vivo. Further experiments were, therefore, devoted to describe mechanisms of glial swelling induced by mediators of secondary brain damage such as glutamate, high K + and lactacidosis.
Excitotoxins: glutamate Glutamate is an excitatory transmitter in the CNS (Fonnum, 1984; Greenamyre, 1986), and, under pathophysiologic conditions, assumes neurotoxic properties (Mayer and Westbrook, 1987). Glutamate is ubiquitous in the central nervous system but strictly compartmentalized in the intracellular space with i.c. concentrations up to 15 mM, more than any other free amino acid in the brain. At least three major post-synaptic receptor types have been identified. They have been named after their primary agonists, NMDA (N-methyl-Daspartate), kainate and quisqualate/AMPA. Despite its role as the major excitatory neurotransmitter, glutamate also acts as a neurotoxin at high extracellular concentrations. Glutamate thus combines physiological excitatory with pathophysiological neurotoxic potencies. To describe this fact, the expression “excitotoxin” has been coined for glutamate (for review, see Mayer and Westbrook, 1987). Extracellular glutamate is rapidly taken up by glial cells against its steep i.c./e.c. concentration gradient (Benjamin and Quastel, 1976; Schousboe et al., 1977; Hertz et al., 1978; Drejer et al., 1982; Ramaharobandro et al., 1982; Barbour et a1.,71988). This process is coupled to a Na+ downhill influx into the cell and thus is energy-dependent. Evidently, the co-uptake of glutamate and
12
sodium may go along with an influx of water, and glial swelling. A significant increase of glial cell volume was indeed shown, first in a preliminary study performed at this laboratory using a high glutamate concentration of 15 mM (Kempski et al., 1982). Similar observations were later reported by Chan and Chu (1989) and Koyama et al. (1991). In a more detailed investigation (Schneider et al., 1992),glutamate was added to C6 cells after a 30 rnin control incubation at concentrations between 50 pM and 10 mM. Astrocytes were exposed to 1 mM glutamate. In all instances glial swelling was observed with maximal increases between 5 % (50 pM glu) and 18% (5 mM glu) of the control volume. At low glutamate concentrations of 50 and 200 pM swelling was only temporary, i.e., cell volume normalized after an initial increase. HPLC measurements demonstrated that the cells were able to remove glutamate from the extracellular space. Glutamate uptake coincided with swelling, and cell volume normalized once all e.c. glutamate had been cleared from the incubation medium. Swelling was Na f -dependent: removal of the e.c./i.c. sodium gradient by a 90 min preincubation with ouabain completely prevented glutamateinduced glial swelling. Based on these observations, the uptake of glutamate together with sodium ions by glial elements resulting in an increase of cellular volume is proposed as one of the mechanisms causing the formation of cytotoxic edema in ischemic and postischemic brain. It should be mentioned, that under pathophysiologic conditions glutamate may reach concentrations in the e.c. space which are well comparable to the highest levels studied here. With tissue concentrations of 12 mM, and a 20% interstitial space, an intracellular concentration of 14 - 15 mM has to be assumed. Therefore, in thevicinity of a traumatic or necrotic focus, where cellular constituents are released to the interstitial space, e.c. glutamate can be expected to temporarily rise even up to 5 - 10 mM, i.e., the higher concentrations used in our in vitro study. 1 mM of glutamate has actually been measured in edema fluid collected from cat brain
with a freezing injury and additional ischemia (Baethmann et al., 1989).
Elevated extracellular K concentrations Extracellular K + concentrations are closely controlled at 3 mM under physiologic conditions. Due to glial uptake and buffering mechanisms extracellular K + levels rarely surpass 12 - 15 mM. Only during pathological events like anoxia, hypoxia, ischemia, or hypoglycemia, and during spreading depression, K + at first increases slowly to a ceiling of 12- 15 mM and then, suddenly, reaches 60 - 80 mM (for review see Walz and Hertz, 1983). During this phase, extensive glial swelling is regularly observed, suggesting high e.c. K concentrations as a mediator mechanism of glial swelling. To study this potential swelling mechanism, the cell suspension in the chamber (Kl = 5 mM) was diluted with isotonic medium of 55 mM K + , keeping the sum of Na+ and K + constant (sodium ions replaced by potassium ions). The final potassium concentration in the suspension was 30 mM. Drugs (furosemide, amiloride, iodoacetate, ouabain) were added 15 min prior to the increase of extracellular potassium and were also present in the high potassium solution. In a subset of experiments anoxia was induced prior to the elevation of e.c. K + . Anoxia was initiated as described above. Exposure of C6 glioma cells to 30 mM K + was followed by very stereotype changes of cell volume: a phase of gradual swelling to a maximum of 108.3 k 0.91 Yo of the control volume after 10 min was followed by slow volume normalization (100.84 k 1.26% after 45 min), although e.c. K + remained elevated during the whole experimental period. Well comparable swelling kinetics were found in astrocytes from primary culture, with a slightly higher swelling maximum of 109.97 k 2.09% after 12 min, and a less rapid and complete normalization of cell volume. Pretreatment with 1.0 mM ouabain or 2.5 mM iodoacetate completely prevented the swelling response. The elevation of e.c. K + during anoxia was followed by earlier swelling of 108.6 rt: 2.68% after already 2 min (P < 0.05 vs. normoxia) and +
+
13
110.2 f 1.85% after 8 min. Iodoacetate prevented the swelling response to K + in anoxia. Amiloride reduced the swelling maximum but did not completely prevent swelling. Furosemide and acetazolamide had even lesser effects. K+-induced glial swelling turns out t o result from an intricate interaction of transport and diffusion processes, and metabolic stimulation - with many open questions remaining. Our concept (Fig. 1) of the major mechanism involved can be summarized as follows: high e.c. K + causes a burst-like stimulation of Na+ /K+-ATPase and, hence, increases the metabolic demands. Oxygen consumption increases and, in addition, lactate is produced. The cell is slightly acidified. In order to maintain a normal i.c. pH, the N a + / H + antiporter extrudes protons and supplies Na+ for further N a + / K + exchange. In addition, K + ions enter the cell via membrane channels or furosemide-inhibitable transport. K + - , C1--, and lactate-ions accumulate intracellularly as the osmotic basis for cell swelling.
rn
K: 4ADPf.P
Activation of GLYCOLYSIS
t
1
I
Naf/Ht-Aniiporter
Anion-Antiporter
n HCO;
Fig. 1. Mechanisms involved in K+-induced glial swelling. High e.c. K + causes a burst-like stimulation of Na+/K+-ATPase activity and of oxygen consumption. In addition, lactate is produced. The cell is acidified, which in turn stimulates N a + / K + exchange to extrude protons. Sodium ions gained may be utilized for further N a + / K + exchange. K + , CI-, and lactate ions accumulate as the osmotic basis for cell swelling. Cell volume normalization involves the export of lactate which may be blocked by quercetin. Additional swelling mechanisms - not incorporated in this figure - include passive potassium influx and furosemide-inhibitable influx mechanisms.
Later, cell volume normalizes slowly, a process involving lactate export, and other, so far unidentified mechanisms. Taken together, the temporary swelling of glia at high K + or glutamate concentrations is the result of homeostatic functions: the maintenance of a constant extracellular potassium concentration, and the removal of e x . glutamate, respectively. The control of the interstitial neuronal environment ranges over glial cell volume control. Acidosis Lactacidosis from anaerobic metabolism is a consequence of cerebral ischemia, seizures and head injury (Siesjo, 1981). A marked decrease of brain tissue pH has been demonstrated in cerebral ischemia particularly in hyperglycemic subjects, where lactic acid may accumulate to 20 - 30 mM and even higher concentrations (Rehncrona et al., 1980; Katsura et al., 1991). Tissue pH may drop down to 5.5 (Chopp et al., 1988). Acidosis has long been suspected as a mediator of brain damage (Siesjo, 1981). Using the in vitro system we were able to confirm the swelling-inducing capacity of extracellular lactacidosis. Again the cells were suspended in physiological medium, which was then rendered acidotic by addition of either sulphuric acid (Kempski et al., 1988a) or lactic acid (Staub et al., 1990). A pH range between 7.6 and 4.2 was studied under strict maintenance of isotonicity. Glial volume was found to increase if the e.c. pH was titrated to 6.8 or below. From this level downward, the extent of swelling depended on the degree of acidosis and the duration of exposure. So, lactacidosis of pH 6.2 for 60 min led to a 24.5% increase in cell volume, while pH 5.0 or 4.2 increased cell size with 51.1% or 90.9%, respectively. Using sulphuric acid for induction of acidosis, swelling was significantly less pronounced. Cell viability was not affected down to pH 6.2. At p H 5.6 cell viability remained normal (89%) for 30 min but then decreased to 73% after 60 min. At pH 4.2 only 21 T ' o of the cells survived 1 h of acidosis. Cell swelling could be inhibited by replacement of Na+ and bicarbonate in the medium by use of
74
choline chloride and HEPES as buffer, suggesting an involvement of Na+/H+ and HCOl/Cl- antiporters in the swelling process. The results enabled us to propose a concept of lactacidosis-induced swelling: addition of lactic acid to bicarbonate buffered media leads to the formation of carbonic acid which immediately dissociates to C 0 2 and water. In turn, intracellular acidosis can develop, since C 0 2 freely passes the cell membrane, and, catalyzed by carbonic anhydrase, again forms carbonic acid. The resulting H + ions are exchanged against sodium ions. As acidosis-induced glial swelling could be partially blocked by SITS and related compounds, an involvement of Cl-/HCOT exchange mechanisms might be involved in addition. A resulting influx of Na+ and C1- ions accompanied by water would explain swelling. Further experiments are required to establish the contribution of other ion transport systems such as Na+/HCOy cotransport.
Conclusions The preservation of a normal cell volume in anoxia even after addition of iodoacetate, or during exposure to ouabain demonstrates that neither the breakdown of energy metabolism, nor inhibition of the active sodium pump suffice to explain cell swelling on the basis of the Donnan equilibrium. Additional mechanisms are required such as alterations of the extracellular homeostasis by accumulation of glutamate or potassium ions, or by the development of lactacidosis. So far these mediator mechanisms have been studied only in separation but it can be assumed that they mutually influence each other, as, e.g., seen from the enhancement of K+-induced swelling in anoxia. As a further conclusion it should be stressed that glial swelling does not indicate glial damage but rather is the result of homeostatic mechanisms designed to protect either the neuronal environment from excess glutamate or K + ions, or the intracellular glial milieu from acidosis.
References Ames, A. and Nesbett, F.B. (1983a) Pathophysiology of ischemic cell death. I. Time of onset of irreversible damage; importance of different compounds of the ischemic insult. Stroke, 14: 219-226. Ames, A. and Nesbett, F.B. (1983b) Pathophysiology of ischemic cell death. 11. Changes of plasma membrane permeability and cell volume. Stroke, 14: 227 - 233. Ames, A. andNesbett, F.B. (1983~)Pathophysiologyof ischemic cell death. 111. Role of extracellular factors. Stroke, 14: 233 - 240. Baethmann, A., Oettinger, W., Rothenfusser, W., Kempski, O., Unterberg, A. and Geiger, R. (1980) Brain edema factors: current state with particular reference to glutamate. Adv. Neurol., 28: 171 - 195. Baethmann, A., Maier-Hauff, K., Schurer, L., Lange, M., Guggenbichler, C., Vogt, W., Jacob, K. and Kempski, 0. (1989) Release of glutamate and free fatty acids in vasogenic brain edema. J. Neurosurg., 70: 578 - 591. Barbour, B., Brew, H. and Attwell, D. (1988) Electrogenic glutamate uptake in glial cells is activated by intracellular potassium. Nature, 335: 433 -435. Benda, P., Lightbody, J., Sato, G., Levine, L. and Sweet, W. (1968) Differentiated rat glial cell strain in tissue culture. Science, 161: 370-371. Benjamin, A.M. and Quastel, J.H. (1976) Cerebral uptakes and exchange diffusion in vitro of L-and D-glutamates. J. Neurochem., 26: 431 -441. Chan, P.H. and Chu, L. (1989) Ketamine protects cultured astrocytes from glutamate-induced swelling. Brain Res., 487: 380-383.
Chopp, M., Welch, K.M.A., Tidwell, C.D. and Helpern, J.A. (1988) Global cerebral ischemia and intracellular pH during hyperglycemia and hypoglycemia in cats. Stroke, 19: 1383- 1387.
Drejer, J., Larsson, O.M. and Schousboe, A. (1982) Characterization of L-glutamate uptake into and release from astrocytes and neurons cultured from different brain regions. Exp. Brain Res., 47: 259 - 269. Fedoroff, S., McAuley, W.A.J., Houle, J.D. and Devon, R.M. (1984) Astrocyte lineage. V. Similarity of astrocytes that form in the presence of dB-CAMP in cultures to reactive astrocytes in vivo. J. Neurosci. Res., 12: 15-27. Fonnum, F. (1984) Glutamate: a neurotransmitter in mammalian brain - short review. J. Neurochem., 42: 1 - 11. Frangakis, M.V. and Kimelberg, H.K.(1984) Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures. Neurochem. Res., 9: 1689- 1698. Greenamyre, J.T. (1986) The role of glutamate in neurotransmissionandinneurologicdisease.Arch. Neurol., 43: 1058 - 1063.
75
Henn, F.A., Goldstein, M.N. and Hamberger A. (1974) Uptake of the neurotransmitter candidate glutamate by glia. Nature, 249: 663 - 664. Hertz, L . , Schousboe, A., Boechler, N., Mukerji, S. and Fedoroff, S. (1978) Kinetic characteristics of the glutamate uptake into normal astrocytes in cultures. Neurochem. Rex, 3: 1 - 14.
Kachel, V. (1976) Basic principles of electrical sizing of cells and particles and their realization in the new instrument “Metricell”. J. Histochem. Cytochem., 24: 21 I - 230. Katsura, K., Ekholm, A., Asplund, F. and Siesjo, B.K. (1991) Extracellular pH in the brain during ischemia: relationship to the severity of lactic acidosis. J. Cereb. Blood Flow Metab., 11: 597-599.
Kempski, O., Gross, U. and Baethmann, A. (1982) An in vitro model of cytotoxic brain edema: cell volume and metabolism of cultivated glial- and nerve-cells. In: W. Driesen, M. Brock and M. Klinger (Eds.), Advances in Neurosurgery, Vol. 10, Springer, Berlin, pp. 254 - 258. Kempski, O., Chaussy, L., Gross, U ., Zimmer, M. and Baethmann, A. (1983) Volume regulation and metabolism of suspended C6 glioma cells: an in vitro model to study cytotoxic brain edema. Brain Res., 279: 217 - 228. Kempski, O., Zimmer, M., Neu, A., von Rosen, F. and Baethmann, A. (1987) Control of glial cell volume in anoxia. Stroke. 18: 623 - 628. Kempski, O., Staub, F., Jansen, M., Schodel, M. and Baethmann, A. (1988a) Glial swelling during extracellular acidosis in vitro. Stroke, 19: 385 - 392. Kempski, O., Staub, F., von Rosen, F., Zimmer, M., Neu, A. and Baethmann, A. (1988b) Molecular mechanisms of glial swelling in vitro. Neurochem. Pathol., 9: 109- 125. Kempski, O., vonRosen, F., Weigt, H., Staub, F., Peters, J. and Baethmann, A. (1991) Glial ion transport and volume control.
Proc. N.Y. Acad. Sci., 633: 306-317. Koyoma, Y., Baba, A. and Iwata, H. (1991) L-Glutamateinduced swelling of cultured astrocytes is dependent on extracellular CaZ+.Neurosci. Lett., 122: 210-212. Lucas, D.R. and Newhouse, J.P. (1957) The toxic effect of sodium L-glutamate on the inner layers of retina. Arch. Ophthalmol., 58: 193 - 204. Macknight, A.D.C. and Leaf, A. (1977) Regulation of cellular volume. Physiol. Rev., 57: 510- 573. Mayer, M.L. and Westbrook, G.L. (1987) Cellular mechanisms underlying excitotoxicity. Trends Neurosci., 10: 59 - 61. Nicklas, W.J. and Browning, E.T. (1983) Glutamate uptakeand metabolism in C-6 glioma cells: alterations by potassium ion and dibutyryl CAMP. J. Neurochem., 41: 179- 187. Ramaharobandro, N., Borg, J., Mandel, P. and Mark, J. (1982) Glutamine and glutamate transport in cultured neuronal and glial cells. Brain Res., 244: 113 - 121. Rehncrona, S., Siesjo, B.K. and Smith, D.S. (1980) Reversible ischemia of the brain: biochemical factors influencing restitution. Aria Physiol. Scand. (Suppl.), 492: 135- 140. Schneider, G.-H., Baethmann, A. and Kempski, 0. (1992) Mechanisms of glial swelling induced by glutamate. Can. J. Physiol. Pharmacol, in press. Schousboe, A., Svenneby, G . and Hertz, L. (1977) Uptake and metabolism in astrocytes cultured from dissociated mouse brain hemispheres. J. Neurochem., 29: 999- 1005. Siesjo, B.K. (1981) Cell damage in the brain: a speculative synthesis. J. Cereb. Blood Flow Metab., 1: 155 - 185. Staub, F., Baethmann, A., Peters, J., Weigt, H. and Kempski, 0. (1990) Effects of lactacidosis on glial cell volume and viability. J. Cereb. Blood Flow Metab., 10: 866- 876. Walz, W. and Hertz, L. (1983) Functional interactions between neurons and astrocytes. 11. Potassium homeostasis at the cellular level. Prog. Neurobiol., 20: 133 - 183.
69
CHAPTER 6
Swelling of C6 glioma cells and astrocytes from glutamate, high K + concentrations or acidosis 0. Kernpskil, F. Staub, G.-H. Schneider, H. Weigt and A. Baethmann
’
Institute f o r Surgical Research, Ludwig-Maximilians University, Klinikum Grosshadern, 0-8000Miinchen 70, and Institute of Neurosurgical Pathophysiology, Johannes Gutenberg-University, 0-6500Mainz, Germany
Introduction
During pathophysiologic events such as ischemia the control of glial cell volume is lost. The swelling of cellular elements in the brain - glial endfeet but also dendrites - is referred to as “cytotoxic brain edema”. Brain edema and the resulting rise in intracranial pressure (ICP) often determine the ultimate outcome of neurosurgical patients. So far, it has been the general understanding that with impaired energy supply, cell swelling will result from the failure of Na+/K+-ATPase according to the pump-leak model of cell volume regulation (Macknight and Leaf, 1977). Glial swelling in cerebral ischemia, however, occurs fast, within 5 min after interruption of the energy supply. In vitro, on the other hand, the onset of swelling depends on the size of the volume of the incubation medium, as elegantly demonstrated by Ames and Nesbett (1983a - c) using the isolated retina. Their observations are best explained by assuming that toxic compounds accumulating in the extracellular environment mediate cell swelling and also nerve cell death. Ames’ data are in line with a variety of other observations which have led to the concept of mediators of secondary brain damage (Baethmann et al., 1980). Mediators are substances released or activated during pathophysiologic events such as ischemia. Excitotoxins (e.g., glutamate), lactic acidosis, free fatty acids, and elevated extracellular
(ex.) concentrations of potassium are the most prominent mediator candidates identified so far. Reports on the occurrence of these mediators under ischemic conditions in vivo are available in abundance. Unfortunately, it is virtually impossible in vivo to discriminate the effects of single mediators on glial volume, since, in addition to multiple interactions, they are released or activated practically simultaneously. In a series of in vitro studies, we have, therefore, analyzed glial cell volume changes during defined and strictly controlled alterations of the extracellular environment in vitro (Kempski et al., 1983, 1987, 1988a,b, 1991; Staub et al., 1990). This article is an attempt to summarize the results. Methods
Glial cell culture Experiments were performed with C6 glioma cells (Benda et al., 1968) and astrocytes from primary culture. The latter were obtained from 3-day-old neonatal rats employing a method adapted from Frangakis and Kimelberg (Frangakis and Kimelberg, 1984; Staub et al., 1990). In brief, forebrains were freed from meninges and choroid plexus under a dissectingmicroscope. The tissue was chopped to fine pieces, and repeatedly digested using dispase. Suspended cells were sedimented and seeded into Petri dishes. The purity of all prepara-
70
tions was assessed by GFAP PAP stains (DAKO). Both cell types were cultured in monolayers in Petri dishes using Dulbecco’s minimal essential medium (DMEM), supplemented with penicillin G (100 IU/ml) and streptomycin (50 pg/ml). Cell growth and differentiation was stimulated by 10% fetal calf serum (FCS). Cells were cultured at 37°C in a humidified atmosphere of 5% CO, and 95% room air. The culture medium was changed and C6 cells were subcultivated daily.
Measurement of cell volume Cell volume was determined by use of a flow cytometer (MetrizellR)combining the Coulter principle with a hydrodynamic focusing technique (Kachel, 1976). The device permits to detect even subtle cell volume changes of 1-2070 with high precision. Latex beads of known diameter served to calibrate the system.
Cell suspension Cells were harvested for the experiments 2 days after subcultivation when the Petri dishes showed confluent monolayers and cells had generated glial processes. This method ensured a fairly constant cell count in the incubation chamber during the various experiments. In primary astrocyte cultures “cell differentiation” was induced 24 h before harvesting by addition of dibutyrylic-cyclic-AMP (dB-CAMP) at a final concentration of 0.5 mM in the medium (Fedoroff et al., 1984). After enzymatic detachment from the Petri dishes with trypsin-EDTA, cells were washed in DMEM + 10% FCS to inactivate trypsin. Then the cells were resuspended, rinsed and centrifuged in FCS-free experimental medium two times.
The experimental mode1 The experimental model permits to quantify cell volume changes at a far higher level of accuracy than possible under pathologic conditions in vivo. Cell volume changes of less than 1% can be detected. Furthermore it is possible to control and maintain important parameters such as ion concentrations, osmolality, pH, PO, and temperature. Suspended C6 cells have a very active oxidative metabolism, as demonstrated by their vivid oxygen consumption (Kempski et al., 1983). The major advantages of using C6 cells are the rapid availability of large cell numbers necessary for reliable flow cytometric volume measurements, and the homogeneous cell size distribution permitting the detection of even subtle volume changes. C6 cells express specific glial markers such as SlOO and GFAP, as well as enzymes, transmitter uptake systems and ion carriers typical for astrocytes (Kempski et al., 1988a). Astrocytes from primary culture are used to verify observations obtained with C6 cells (Kempski et al., 1988a, 1991; Staub et al., 1990; Schneider et al., 1992). So far, astrocytes have always mirrored the observations previously made with C6 cells. It is concluded that the basic mechanisms of cell volume control are very similar for both cell types.
Incubation chamber After the second centrifugation cells were resuspended and quickly introduced into a plexiglass incubation chamber (Kempski et al., 1983). Important extracellular parameters like temperature (37”C), pH and PO, were monitored by electrodes in the chamber thus allowing to keep them constant. A membrane oxygenator from permeable silicon rubber tubing maintains constant partial pressures of 0,, CO, and N,. The medium was buffered with bicarbonate (24 mM), and extracellular pH was kept at 7.4 by regulating the flow of CO, accordingly. A teflon-covered magnetic stirrer in the chamber prevented sedimentation of the suspended cells. Samples of the suspension were retrieved or drugs added through openings in the chamber wall.
Results and discussion
Anoxia The in vitro model was first used to evaluate whether anoxia or inhibition of the cellular energy supply without any further changes of the extracellular environment would be accompanied by swelling (Kempski et al., 1987).Anoxia was induced by discontinuation of the oxygen supply to the mem-
71
brane oxygenator, and subsequent ventilation with nitrogen. In addition the incubation chamber was placed into an air-tight tent flooded with nitrogen to avoid any leakage of oxygen into the chamber. Low oxygen tensions possibly remaining in the medium were consumed by the respiration of the dense cell suspension within minutes. Anoxia was maintained for 2 h - and cell swelling was never observed. The increase of e.c. lactate indicated that anaerobic glycolysis was sufficient to maintain ion gradients and cell volume control. Indeed the analysis of i.c. electrolyte concentrations by atomic absorption spectrometry revealed normal i.c. Na+ concentrations. Therefore, iodoacetate was used to inhibit glycolysis in addition to anoxia. This treatment successfully abolished the i.c./e.c. Na+ gradient within 120 min, but, again, cell volume was unaffected (Kempski et al., 1987). These results are in line with observations of Ames and Nesbett using the isolated retina (1983a - c). In anoxia retinal swelling was absent if the extracellular incubation medium was large enough to dilute toxic material released from the tissue. Medium conditioned by anoxic retinas, or therestriction of the e.c. fluid volume during anoxia was able to enhance tissue swelling. We conclude that in the narrow, restricted e.c. space of the brain in vivo, mediators of secondary brain damage released or activated by pathophysiologic events accumulate to cause glial swelling and, eventually, glial and nerve cell death.
Inhibition of Na+/K+-ATPase by ouabain In another study this conclusion found further support. Here, the sodium-potassium pump was blocked by the cardiac glycoside ouabain. The Na+ gradient was lost by this treatment within 90 min in C6 cells (Kempski et al., 1988b). Intracellular Na+ measurements did demonstrate a near six-fold increase of intracellular Na+ content from a low 13 x mol/cell to 78 x mol/cell, and, hence, proved that the Na+/K+-ATPase was effectively inhibited by 1 mM ouabain. Simultaneously asteep drop in the i.c. potassium content from 190 x mol/cell to 30 x mol/cell was
observed. Forty-five minutes of ouabain treatment were not sufficient to completely collapse the sodium gradient: i.c. Na+ was still 56 x mol/cell. Just as observed with complete anoxia (Kempski et al., 1987), the inhibition of the Na+/K+ pump did not cause cell swelling (Kempski et al., 1988b). Again, the data support the contention that failure of the energy supply per se, or of sodium transport by Na+/K+-ATPase do not suffice to induce glial swelling in vivo. Further experiments were, therefore, devoted to describe mechanisms of glial swelling induced by mediators of secondary brain damage such as glutamate, high K + and lactacidosis.
Excitotoxins: glutamate Glutamate is an excitatory transmitter in the CNS (Fonnum, 1984; Greenamyre, 1986), and, under pathophysiologic conditions, assumes neurotoxic properties (Mayer and Westbrook, 1987). Glutamate is ubiquitous in the central nervous system but strictly compartmentalized in the intracellular space with i.c. concentrations up to 15 mM, more than any other free amino acid in the brain. At least three major post-synaptic receptor types have been identified. They have been named after their primary agonists, NMDA (N-methyl-Daspartate), kainate and quisqualate/AMPA. Despite its role as the major excitatory neurotransmitter, glutamate also acts as a neurotoxin at high extracellular concentrations. Glutamate thus combines physiological excitatory with pathophysiological neurotoxic potencies. To describe this fact, the expression “excitotoxin” has been coined for glutamate (for review, see Mayer and Westbrook, 1987). Extracellular glutamate is rapidly taken up by glial cells against its steep i.c./e.c. concentration gradient (Benjamin and Quastel, 1976; Schousboe et al., 1977; Hertz et al., 1978; Drejer et al., 1982; Ramaharobandro et al., 1982; Barbour et a1.,71988). This process is coupled to a Na+ downhill influx into the cell and thus is energy-dependent. Evidently, the co-uptake of glutamate and
12
sodium may go along with an influx of water, and glial swelling. A significant increase of glial cell volume was indeed shown, first in a preliminary study performed at this laboratory using a high glutamate concentration of 15 mM (Kempski et al., 1982). Similar observations were later reported by Chan and Chu (1989) and Koyama et al. (1991). In a more detailed investigation (Schneider et al., 1992),glutamate was added to C6 cells after a 30 rnin control incubation at concentrations between 50 pM and 10 mM. Astrocytes were exposed to 1 mM glutamate. In all instances glial swelling was observed with maximal increases between 5 % (50 pM glu) and 18% (5 mM glu) of the control volume. At low glutamate concentrations of 50 and 200 pM swelling was only temporary, i.e., cell volume normalized after an initial increase. HPLC measurements demonstrated that the cells were able to remove glutamate from the extracellular space. Glutamate uptake coincided with swelling, and cell volume normalized once all e.c. glutamate had been cleared from the incubation medium. Swelling was Na f -dependent: removal of the e.c./i.c. sodium gradient by a 90 min preincubation with ouabain completely prevented glutamateinduced glial swelling. Based on these observations, the uptake of glutamate together with sodium ions by glial elements resulting in an increase of cellular volume is proposed as one of the mechanisms causing the formation of cytotoxic edema in ischemic and postischemic brain. It should be mentioned, that under pathophysiologic conditions glutamate may reach concentrations in the e.c. space which are well comparable to the highest levels studied here. With tissue concentrations of 12 mM, and a 20% interstitial space, an intracellular concentration of 14 - 15 mM has to be assumed. Therefore, in thevicinity of a traumatic or necrotic focus, where cellular constituents are released to the interstitial space, e.c. glutamate can be expected to temporarily rise even up to 5 - 10 mM, i.e., the higher concentrations used in our in vitro study. 1 mM of glutamate has actually been measured in edema fluid collected from cat brain
with a freezing injury and additional ischemia (Baethmann et al., 1989).
Elevated extracellular K concentrations Extracellular K + concentrations are closely controlled at 3 mM under physiologic conditions. Due to glial uptake and buffering mechanisms extracellular K + levels rarely surpass 12 - 15 mM. Only during pathological events like anoxia, hypoxia, ischemia, or hypoglycemia, and during spreading depression, K + at first increases slowly to a ceiling of 12- 15 mM and then, suddenly, reaches 60 - 80 mM (for review see Walz and Hertz, 1983). During this phase, extensive glial swelling is regularly observed, suggesting high e.c. K concentrations as a mediator mechanism of glial swelling. To study this potential swelling mechanism, the cell suspension in the chamber (Kl = 5 mM) was diluted with isotonic medium of 55 mM K + , keeping the sum of Na+ and K + constant (sodium ions replaced by potassium ions). The final potassium concentration in the suspension was 30 mM. Drugs (furosemide, amiloride, iodoacetate, ouabain) were added 15 min prior to the increase of extracellular potassium and were also present in the high potassium solution. In a subset of experiments anoxia was induced prior to the elevation of e.c. K + . Anoxia was initiated as described above. Exposure of C6 glioma cells to 30 mM K + was followed by very stereotype changes of cell volume: a phase of gradual swelling to a maximum of 108.3 k 0.91 Yo of the control volume after 10 min was followed by slow volume normalization (100.84 k 1.26% after 45 min), although e.c. K + remained elevated during the whole experimental period. Well comparable swelling kinetics were found in astrocytes from primary culture, with a slightly higher swelling maximum of 109.97 k 2.09% after 12 min, and a less rapid and complete normalization of cell volume. Pretreatment with 1.0 mM ouabain or 2.5 mM iodoacetate completely prevented the swelling response. The elevation of e.c. K + during anoxia was followed by earlier swelling of 108.6 rt: 2.68% after already 2 min (P < 0.05 vs. normoxia) and +
+
13
110.2 f 1.85% after 8 min. Iodoacetate prevented the swelling response to K + in anoxia. Amiloride reduced the swelling maximum but did not completely prevent swelling. Furosemide and acetazolamide had even lesser effects. K+-induced glial swelling turns out t o result from an intricate interaction of transport and diffusion processes, and metabolic stimulation - with many open questions remaining. Our concept (Fig. 1) of the major mechanism involved can be summarized as follows: high e.c. K + causes a burst-like stimulation of Na+ /K+-ATPase and, hence, increases the metabolic demands. Oxygen consumption increases and, in addition, lactate is produced. The cell is slightly acidified. In order to maintain a normal i.c. pH, the N a + / H + antiporter extrudes protons and supplies Na+ for further N a + / K + exchange. In addition, K + ions enter the cell via membrane channels or furosemide-inhibitable transport. K + - , C1--, and lactate-ions accumulate intracellularly as the osmotic basis for cell swelling.
rn
K: 4ADPf.P
Activation of GLYCOLYSIS
t
1
I
Naf/Ht-Aniiporter
Anion-Antiporter
n HCO;
Fig. 1. Mechanisms involved in K+-induced glial swelling. High e.c. K + causes a burst-like stimulation of Na+/K+-ATPase activity and of oxygen consumption. In addition, lactate is produced. The cell is acidified, which in turn stimulates N a + / K + exchange to extrude protons. Sodium ions gained may be utilized for further N a + / K + exchange. K + , CI-, and lactate ions accumulate as the osmotic basis for cell swelling. Cell volume normalization involves the export of lactate which may be blocked by quercetin. Additional swelling mechanisms - not incorporated in this figure - include passive potassium influx and furosemide-inhibitable influx mechanisms.
Later, cell volume normalizes slowly, a process involving lactate export, and other, so far unidentified mechanisms. Taken together, the temporary swelling of glia at high K + or glutamate concentrations is the result of homeostatic functions: the maintenance of a constant extracellular potassium concentration, and the removal of e x . glutamate, respectively. The control of the interstitial neuronal environment ranges over glial cell volume control. Acidosis Lactacidosis from anaerobic metabolism is a consequence of cerebral ischemia, seizures and head injury (Siesjo, 1981). A marked decrease of brain tissue pH has been demonstrated in cerebral ischemia particularly in hyperglycemic subjects, where lactic acid may accumulate to 20 - 30 mM and even higher concentrations (Rehncrona et al., 1980; Katsura et al., 1991). Tissue pH may drop down to 5.5 (Chopp et al., 1988). Acidosis has long been suspected as a mediator of brain damage (Siesjo, 1981). Using the in vitro system we were able to confirm the swelling-inducing capacity of extracellular lactacidosis. Again the cells were suspended in physiological medium, which was then rendered acidotic by addition of either sulphuric acid (Kempski et al., 1988a) or lactic acid (Staub et al., 1990). A pH range between 7.6 and 4.2 was studied under strict maintenance of isotonicity. Glial volume was found to increase if the e.c. pH was titrated to 6.8 or below. From this level downward, the extent of swelling depended on the degree of acidosis and the duration of exposure. So, lactacidosis of pH 6.2 for 60 min led to a 24.5% increase in cell volume, while pH 5.0 or 4.2 increased cell size with 51.1% or 90.9%, respectively. Using sulphuric acid for induction of acidosis, swelling was significantly less pronounced. Cell viability was not affected down to pH 6.2. At p H 5.6 cell viability remained normal (89%) for 30 min but then decreased to 73% after 60 min. At pH 4.2 only 21 T ' o of the cells survived 1 h of acidosis. Cell swelling could be inhibited by replacement of Na+ and bicarbonate in the medium by use of
74
choline chloride and HEPES as buffer, suggesting an involvement of Na+/H+ and HCOl/Cl- antiporters in the swelling process. The results enabled us to propose a concept of lactacidosis-induced swelling: addition of lactic acid to bicarbonate buffered media leads to the formation of carbonic acid which immediately dissociates to C 0 2 and water. In turn, intracellular acidosis can develop, since C 0 2 freely passes the cell membrane, and, catalyzed by carbonic anhydrase, again forms carbonic acid. The resulting H + ions are exchanged against sodium ions. As acidosis-induced glial swelling could be partially blocked by SITS and related compounds, an involvement of Cl-/HCOT exchange mechanisms might be involved in addition. A resulting influx of Na+ and C1- ions accompanied by water would explain swelling. Further experiments are required to establish the contribution of other ion transport systems such as Na+/HCOy cotransport.
Conclusions The preservation of a normal cell volume in anoxia even after addition of iodoacetate, or during exposure to ouabain demonstrates that neither the breakdown of energy metabolism, nor inhibition of the active sodium pump suffice to explain cell swelling on the basis of the Donnan equilibrium. Additional mechanisms are required such as alterations of the extracellular homeostasis by accumulation of glutamate or potassium ions, or by the development of lactacidosis. So far these mediator mechanisms have been studied only in separation but it can be assumed that they mutually influence each other, as, e.g., seen from the enhancement of K+-induced swelling in anoxia. As a further conclusion it should be stressed that glial swelling does not indicate glial damage but rather is the result of homeostatic mechanisms designed to protect either the neuronal environment from excess glutamate or K + ions, or the intracellular glial milieu from acidosis.
References Ames, A. and Nesbett, F.B. (1983a) Pathophysiology of ischemic cell death. I. Time of onset of irreversible damage; importance of different compounds of the ischemic insult. Stroke, 14: 219-226. Ames, A. and Nesbett, F.B. (1983b) Pathophysiology of ischemic cell death. 11. Changes of plasma membrane permeability and cell volume. Stroke, 14: 227 - 233. Ames, A. andNesbett, F.B. (1983~)Pathophysiologyof ischemic cell death. 111. Role of extracellular factors. Stroke, 14: 233 - 240. Baethmann, A., Oettinger, W., Rothenfusser, W., Kempski, O., Unterberg, A. and Geiger, R. (1980) Brain edema factors: current state with particular reference to glutamate. Adv. Neurol., 28: 171 - 195. Baethmann, A., Maier-Hauff, K., Schurer, L., Lange, M., Guggenbichler, C., Vogt, W., Jacob, K. and Kempski, 0. (1989) Release of glutamate and free fatty acids in vasogenic brain edema. J. Neurosurg., 70: 578 - 591. Barbour, B., Brew, H. and Attwell, D. (1988) Electrogenic glutamate uptake in glial cells is activated by intracellular potassium. Nature, 335: 433 -435. Benda, P., Lightbody, J., Sato, G., Levine, L. and Sweet, W. (1968) Differentiated rat glial cell strain in tissue culture. Science, 161: 370-371. Benjamin, A.M. and Quastel, J.H. (1976) Cerebral uptakes and exchange diffusion in vitro of L-and D-glutamates. J. Neurochem., 26: 431 -441. Chan, P.H. and Chu, L. (1989) Ketamine protects cultured astrocytes from glutamate-induced swelling. Brain Res., 487: 380-383.
Chopp, M., Welch, K.M.A., Tidwell, C.D. and Helpern, J.A. (1988) Global cerebral ischemia and intracellular pH during hyperglycemia and hypoglycemia in cats. Stroke, 19: 1383- 1387.
Drejer, J., Larsson, O.M. and Schousboe, A. (1982) Characterization of L-glutamate uptake into and release from astrocytes and neurons cultured from different brain regions. Exp. Brain Res., 47: 259 - 269. Fedoroff, S., McAuley, W.A.J., Houle, J.D. and Devon, R.M. (1984) Astrocyte lineage. V. Similarity of astrocytes that form in the presence of dB-CAMP in cultures to reactive astrocytes in vivo. J. Neurosci. Res., 12: 15-27. Fonnum, F. (1984) Glutamate: a neurotransmitter in mammalian brain - short review. J. Neurochem., 42: 1 - 11. Frangakis, M.V. and Kimelberg, H.K.(1984) Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures. Neurochem. Res., 9: 1689- 1698. Greenamyre, J.T. (1986) The role of glutamate in neurotransmissionandinneurologicdisease.Arch. Neurol., 43: 1058 - 1063.
75
Henn, F.A., Goldstein, M.N. and Hamberger A. (1974) Uptake of the neurotransmitter candidate glutamate by glia. Nature, 249: 663 - 664. Hertz, L . , Schousboe, A., Boechler, N., Mukerji, S. and Fedoroff, S. (1978) Kinetic characteristics of the glutamate uptake into normal astrocytes in cultures. Neurochem. Rex, 3: 1 - 14.
Kachel, V. (1976) Basic principles of electrical sizing of cells and particles and their realization in the new instrument “Metricell”. J. Histochem. Cytochem., 24: 21 I - 230. Katsura, K., Ekholm, A., Asplund, F. and Siesjo, B.K. (1991) Extracellular pH in the brain during ischemia: relationship to the severity of lactic acidosis. J. Cereb. Blood Flow Metab., 11: 597-599.
Kempski, O., Gross, U. and Baethmann, A. (1982) An in vitro model of cytotoxic brain edema: cell volume and metabolism of cultivated glial- and nerve-cells. In: W. Driesen, M. Brock and M. Klinger (Eds.), Advances in Neurosurgery, Vol. 10, Springer, Berlin, pp. 254 - 258. Kempski, O., Chaussy, L., Gross, U ., Zimmer, M. and Baethmann, A. (1983) Volume regulation and metabolism of suspended C6 glioma cells: an in vitro model to study cytotoxic brain edema. Brain Res., 279: 217 - 228. Kempski, O., Zimmer, M., Neu, A., von Rosen, F. and Baethmann, A. (1987) Control of glial cell volume in anoxia. Stroke. 18: 623 - 628. Kempski, O., Staub, F., Jansen, M., Schodel, M. and Baethmann, A. (1988a) Glial swelling during extracellular acidosis in vitro. Stroke, 19: 385 - 392. Kempski, O., Staub, F., von Rosen, F., Zimmer, M., Neu, A. and Baethmann, A. (1988b) Molecular mechanisms of glial swelling in vitro. Neurochem. Pathol., 9: 109- 125. Kempski, O., vonRosen, F., Weigt, H., Staub, F., Peters, J. and Baethmann, A. (1991) Glial ion transport and volume control.
Proc. N.Y. Acad. Sci., 633: 306-317. Koyoma, Y., Baba, A. and Iwata, H. (1991) L-Glutamateinduced swelling of cultured astrocytes is dependent on extracellular CaZ+.Neurosci. Lett., 122: 210-212. Lucas, D.R. and Newhouse, J.P. (1957) The toxic effect of sodium L-glutamate on the inner layers of retina. Arch. Ophthalmol., 58: 193 - 204. Macknight, A.D.C. and Leaf, A. (1977) Regulation of cellular volume. Physiol. Rev., 57: 510- 573. Mayer, M.L. and Westbrook, G.L. (1987) Cellular mechanisms underlying excitotoxicity. Trends Neurosci., 10: 59 - 61. Nicklas, W.J. and Browning, E.T. (1983) Glutamate uptakeand metabolism in C-6 glioma cells: alterations by potassium ion and dibutyryl CAMP. J. Neurochem., 41: 179- 187. Ramaharobandro, N., Borg, J., Mandel, P. and Mark, J. (1982) Glutamine and glutamate transport in cultured neuronal and glial cells. Brain Res., 244: 113 - 121. Rehncrona, S., Siesjo, B.K. and Smith, D.S. (1980) Reversible ischemia of the brain: biochemical factors influencing restitution. Aria Physiol. Scand. (Suppl.), 492: 135- 140. Schneider, G.-H., Baethmann, A. and Kempski, 0. (1992) Mechanisms of glial swelling induced by glutamate. Can. J. Physiol. Pharmacol, in press. Schousboe, A., Svenneby, G . and Hertz, L. (1977) Uptake and metabolism in astrocytes cultured from dissociated mouse brain hemispheres. J. Neurochem., 29: 999- 1005. Siesjo, B.K. (1981) Cell damage in the brain: a speculative synthesis. J. Cereb. Blood Flow Metab., 1: 155 - 185. Staub, F., Baethmann, A., Peters, J., Weigt, H. and Kempski, 0. (1990) Effects of lactacidosis on glial cell volume and viability. J. Cereb. Blood Flow Metab., 10: 866- 876. Walz, W. and Hertz, L. (1983) Functional interactions between neurons and astrocytes. 11. Potassium homeostasis at the cellular level. Prog. Neurobiol., 20: 133 - 183.
