Volume regulation and metabolism of suspended C6 glioma cells: An in vitro model to study cytotoxic brain edema

Kempski, Oliver; Chaussy, L.; Groß, U.; Zimmer, Michael; Baethmann, A.

doi:10.1016/0006-8993(83)90180-4

Cited by 93 publications

(27 citation statements)

References 29 publications

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“…Reduction of the extracellular osmolality from 300 to 160 mosm/1 resulted in immediate swelling of the C6 glioma cells (Figure 4) followed by complete volume normalization. As in previous studies 7 cell volume returned to the control level within 30-60 minutes, although the glial cells remained exposed to the hypotonic medium. Neither anoxia nor iodoacetate alone inhibited cell volume normalization after immediate swelling during the hyposmotic suspension.…”

Section: Resultssupporting

confidence: 79%

“…Previous experiments have shown that C6 glioma cells spontaneously recover normal cell volume after initial swelling during exposure to a hypotonic medium. 7 The results of this study suggest that interruption of the energy supply alone is not sufficient to induce glial swelling within a period comparable to in vivo conditions. The findings strongly support the hypothesis that cytotoxic cell swelling in ischemia involves additional factors, such as neurotoxic mediator compounds.…”

mentioning

confidence: 67%

“…The cells were harvested and suspended in serum-free medium as previously described. 7 EXPERIMENTAL SETUP. The cell suspension was immediately transferred to a plexiglass incubation chamber which controlled pH, Po 2 , and temperature using respective electrodes.…”

Section: Control Of Glial Cell Volume In Anoxiamentioning

confidence: 99%

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Control of glial cell volume in anoxia. In vitro studies on ischemic cell swelling.

Kempski¹,

Zimmer²,

Neu³

et al. 1987

Stroke

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Volume regulation of C6 glial cells was studied in anoxia in vitro to improve the understanding of ischemic cell swelling in the brain. Contrary to in vivo conditions, anoxia or anoxia plus iodoacetate for additional inhibition of anaerobic energy metabolism did not induce glial swelling. However, intracellular K + was markedly decreased while intracellular Na + increased. Induction of energy failure by anoxia plus iodoacetate was found to prevent the regulatory volume decrease on hyposmotic exposure of the cells, which is regularly observed hi normoxic control conditions. Hyposmotic exposure hi anoxia plus iodoacetate led only to an initial tendency of cell volume normalization followed by secondary cell swelling. This was associated with a net increase of intracellular Na + that may explain the failure of volume regulation under these circumstances. Maintenance of a normal glial cell size during complete energy deprivation by anoxia plus iodoacetate hi isotonic medium strongly indicates that energy failure per se does not suffice to induce cell swelling. Cell swelling hi cerebral ischemia in vivo thus is likely to require additional mechanisms, most likely an Increase of membrane permeability to Na + , which may be caused by release and accumulation of excitotoxins such as glutamate or by an extracellular release of K + . Such a mechanism would hardly influence the extracellular homeostasis in vitro due to the large medium-to-cell volume ratio. The findings demonstrate, nonetheless, the significance of a competent energy metabolism to support cell volume regulation. This is concluded from the failure of regulatory volume decrease of hypotonlcally suspended glial cells hi anoxia plus iodoacetate. The experimental conditions employed appear particularly useful to analyze and to differentiate on a molecular level the complex interactions resulting hi ischemic cell swelling hi the brain. {Stroke 1987;18:623-628) W hen the cerebral circulation is interrupted or decreases below a critical threshold, drastic swelling of dendrites and glial cells ensues. A shift of fluid and Na + from the extracellular to the intracellular space is seen after a latency of only 2-3 minutes.1 " 4 The resulting energy failure is thought to disturb the double Donnan equilibrium, which under physiologic conditions ensures a stable cell volume.5 This interpretation, however, might be too simple. The rapid onset of swelling and the complete breakdown of ion gradients suggest an involvement of additional pathogenetic mechanisms, e.g., release or activation of mediator compounds 6 that promote the development of cytotoxic brain edema by increasing membrane permeability or by inflicting membrane damage.Numerous in vivo studies have been performed to elucidate ischemic cell swelling in the brain. The multitude of parameters changing simultaneously, however, make it difficult to determine the significance of individual factors. Therefore, an in vitro method has been established 7 to study the specific processes causing cell swelling and eventuall...

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Section: Resultssupporting

confidence: 79%

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confidence: 67%

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Control of glial cell volume in anoxia. In vitro studies on ischemic cell swelling.

Kempski¹,

Zimmer²,

Neu³

et al. 1987

Stroke

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“…A magnetic stirrer prevented sedimen tation. Further details have been published (Kempski et a!., 1983(Kempski et a!., , 1988.…”

Section: Cell Culturementioning

confidence: 99%

Swelling, Acidosis, and Irreversible Damage of Glial Cells from Exposure to Arachidonic Acid in vitro

Staub¹,

Winkler²,

Peters³

et al. 1994

J Cereb Blood Flow Metab

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Swelling and damage of C6 glioma cells and of primary cultured astrocytes were analyzed in vitro during incubation with arachidonic acid (AA; 20:4). The cells were suspended in a physiological medium supplemented with AA at concentrations of 0.001–1.0 m M. Cell swelling was quantified by flow cytometry with hydrodynamic focusing. Flow cytometry was also utilized for assessment of cell viability by exclusion of the fluorescent dye propidium iodide and for measurement of the intracellular pH (pHi) by 2′,7′-bis-(2-carboxyethyl)−5(and −6)carboxyfluorescein. Administration of AA caused an immediate dose-dependent swelling of C6 glioma cells, even at a concentration of 0.01 m M. At this level cell volume increased within 20 min to 105.0% of control, at 0.1 m M to 111.0%, while at 1.0 m M to 123.7%. Following a phase of rapid cell volume increase, swelling leveled off during the subsequent observation period of 70 min. Viability of the C6 glioma cells was 90% under control conditions. It remained unchanged after raising AA concentrations to 0.1 m M. At 0.5 m M, however, cell viability fell to 72.8%, and at 1.0 m M to 32.7%. pHi of the glioma cells was 7.3 under control conditions. In parallel with the early swelling phase, AA led to a dose-dependent decrease of the intracellular pH and an elevated lactate production of the cells. During incubation with 0.1 m M AA, pHi decreased to 7.06 after 5 min, but recovered to normal subsequently. In addition, swelling-inducing properties of linoleic (18:2) or stearic (18:0) acid were analyzed for evaluation of the specificity of glial swelling induced by AA. Whereas stearic acid (0.1 m M) failed to induce a swelling response, linoleic acid (0.1 m M) was found to be effective. The volume increase of the glial cells, however, was only half of that found during exposure to AA at the same concentration. Further, glial swelling from AA or linoleic acid was completely inhibited by the aminosteroid U-74389F, an antagonist of lipid peroxidation. Finally, omission of Na+ ions in the suspension medium with replacement by choline led also to inhibition of the cell volume increase by AA. Experiments using astrocytes from primary culture confirmed the swelling-inducing properties of AA at a quantitative level, whereas vulnerability of the cells to AA was increased. The present results demonstrate an important role of AA in cytotoxic swelling and irreversible damage of glial cells at concentrations that occur in vivo in cerebral ischemia or trauma. The damaging potential of AA might be enhanced by a concurrently evolving intracellular acidosis, stimulating the formation of oxygen-derived free radicals and lipid peroxidation.

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“…Astrocytes in vitro have been shown to exhibit RVD after swelling in hypotonic media (Kempski et al, 1983;Kimelberg and Frangakis, 1985;Olson et al, 1986) which is associated with an increased permeability to mannitol (Kimelberg and Goderie, 1988) reversible membrane depolarization (Kimelberg and O'Connor, 1988) and release of 3H-taurine (Pasantes-Morales and Schousboe, 1988).…”

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confidence: 99%

Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures

et al. 1990

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Volume regulation and metabolism of suspended C6 glioma cells: An in vitro model to study cytotoxic brain edema

Cited by 93 publications

References 29 publications

Control of glial cell volume in anoxia. In vitro studies on ischemic cell swelling.

Control of glial cell volume in anoxia. In vitro studies on ischemic cell swelling.

Swelling, Acidosis, and Irreversible Damage of Glial Cells from Exposure to Arachidonic Acid in vitro

Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures

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