Effect of hypothermia on the volume of rat glial cells

Abstract

1. The cell volume of suspended C6 glioma cells and primary cultured rat astrocytes was measured at normothermia (37 degrees C), and at mild (32 degrees C) and moderate (27 degrees C) hypothermia by flow cytometry with electrical cell sizing. 2. Under control conditions (37 degrees C), C6 glioma cells had a volume of 809 +/- 29 microm3. Moderate hypothermia (27 degrees C) led to rapid cell swelling, with a maximum volume of 113.1 +/- 1.3 % of control being achieved after 50 min. After rewarming to 37 degrees C, cell volume recovered very slowly and incompletely (to 107.2 +/- 0.4 % of control). Less severe hypothermia (32 degrees C) led to a smaller increase in cell volume (108.7 +/- 0.5 % of control). 3. The maximal cell swelling response and the kinetics of swelling were similar in C6 glioma cells and primary cultured astrocytes. 4. Hypothermia-induced cell swelling was dependent on the presence of extracellular Na+ and was reduced by the Na+-H+ antiporter inhibitor EIPA. 5. The underlying mechanisms of hypothermia-induced cell swelling are an intracellular accumulation of Na+ by (1) differential effects of hypothermia on the membrane permeabilities of Na+ and K+ and (2) activation of the Na+-H+ antiporter by a shift of its activation curve to a more alkaline value.

Figure 1. Volume response of C6 glioma cells at different levels of hypothermia

The cell volume of suspended cells was measured by flow cytometry with electrical cell sizing. The cell volume response of the cells is given as a percentage of the cell volume under baseline conditions, which was set at 100 % (means ± s.e.m.; n = 5). Under baseline conditions, the cells had an absolute volume of 809 ± 29 μm³. Whereas at 37 °C no increase in the cell volume occurred, a significant swelling response developed during hypothermia. Cell swelling due to hypothermia was only partially reversible upon restoration of the ambient temperature to 37 °C. *P < 0.05vs. 37 °C (Control); †P < 0.05vs. 32 °C.

Figure 2. Correlation of the cell volume response and the degree of hypothermia

The maximal cell volume response (at 50 min) obtained from each experiment is plotted against the degree of hypothermia applied. The Spearman correlation coefficient (r_s = −0.90, P < 0.001, n = 20) demonstrates the close relationship between the two parameters.

Figure 3. Cell volume response of primary cultured astrocytes to hypothermia of 32 °C

Exposure of primary cultured astrocytes to hypothermia (32 °C) also elicited cell swelling of comparable intensity to that observed in C6 glioma cells (cf. Fig. 1). As for the C6 glioma cells, cell volume recovered slowly following a return to 37 °C. Values are means ± s.e.m. (n = 5). *P < 0.05vs. baseline at 37 °C.

Figure 4. Influence of Na⁺-free medium on hypothermia-induced cell swelling (32 °C)

Omission of Na⁺ from the suspension medium abolished hypothermia-induced cell swelling in C6 glioma cells completely. Values are means ± s.e.m. (n = 5). *P < 0.008vs. Control.

Figure 5. Influence of Cl⁻-free medium on hypothermia-induced cell swelling (32 °C)

Omission of Cl⁻ from the suspension medium had no significant influence on hypothermia-induced swelling in C6 glioma cells. Values are means ± s.e.m. (n = 5).

Figure 6. Inhibition of the Na⁺–H⁺ antiporter during hypothermia-induced cell swelling (32 °C)

Inhibition of the Na⁺–H⁺ antiporter in C6 glioma cells by EIPA (50 μM) significantly reduced hypothermia-induced cell swelling by ≈45 %. Cell volume recovery during rewarming of the cells to 37 °C was complete after this treatment. Values are means ± s.e.m. (n = 5). *P < 0.016vs. Control.

Figure 7. Schematic drawing of the mechanisms responsible for hypothermia-induced cell swelling

Hypothermia and ouabain both inhibit the Na⁺,K⁺-ATPase. In the case of ouabain, the cells maintain their cell volume and their membrane potential because the subsequent compensatory Na⁺ influx and K⁺ efflux occur in a 1:1 ratio. Hypothermia changes this ratio in favour of the influx of Na⁺. The evolving intracellular Na⁺ accumulation leads to cell swelling. In addition, hypothermia activates the Na⁺–H⁺ exchanger leading to additional influx of Na⁺.