Effect of hypothermia on the volume of rat glial cells

The cell volume of suspended C6 glioma cells and primary cultured rat astrocytes was measured at normothermia (37 °C), and at mild (32 °C) and moderate (27 °C) hypothermia by flow cytometry with electrical cell sizing. Under control conditions (37 °C), C6 glioma cells had a volume of 809 ± 29 μm3. Moderate hypothermia (27 °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 °C, cell volume recovered very slowly and incompletely (to 107.2 ± 0.4 % of control). Less severe hypothermia (32 °C) led to a smaller increase in cell volume (108.7 ± 0.5 % of control). The maximal cell swelling response and the kinetics of swelling were similar in C6 glioma cells and primary cultured astrocytes. Hypothermia-induced cell swelling was dependent on the presence of extracellular Na+ and was reduced by the Na+–H+ antiporter inhibitor EIPA. 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. The maintenance of a normal cell volume is indispensable for proper cell function and survival (Macknight, 1987). Under physiological conditions, the cell volume is kept constant within a narrow range by a variety of highly efficient and well characterised regulatory mechanisms, including the Na+–H+ antiporter (Rotin & Grinstein, 1989), the K+-Cl− cotransporter (Hallows & Knauf, 1994), the Na+-K+-2Cl− cotransporter (Tas et al. 1987) and the Cl−-HCO3− exchanger (Kimelberg et al. 1979) (for a review see Strange, 1994). However, during various pathological circumstances these mechanisms are overwhelmed, and alterations of the cell volume, most often cell swelling, are observed. One example of such a pathological condition is deep hypothermia of the brain. From experiments performed by Brendel and colleagues in the 1960s (Thauer & Brendel, 1962), it is known that deep brain hypothermia of less than 10°C induces massive, even fatal, swelling of the brain. This swelling was associated with the accumulation of Na+ in the brain parenchyma. However, additional pathophysiological details were unknown at that time as further studies at the molecular and cellular level were not performed. Scientific interest in hypothermia resurfaced after it was shown that not only deep hypothermia (10–15°C), as has been known for a long time, but also mild hypothermia (∼32°C), affords neuroprotection after cerebral ischaemia and trauma (Morikawa et al. 1992). This finding has far reaching clinical consequences, since mild hypothermia has the advantage of being associated with fewer and less serious side effects than deep hypothermia (Ginsberg et al. 1992). However, it is not known whether, in addition to its neuroprotective effect, mild hypothermia also leads to swelling of brain tissue, as seen during deep hypothermia. Hypothermia-induced brain tissue swelling could partially mask positive effects of mild hypothermia on the injured brain. We have therefore investigated the effect of mild (32°C) to moderate (27°C) hypothermia on the cell volume of glial cells, as these cells appear to be specifically involved in the swelling in the brain after trauma and ischaemia (Gerschenfeld et al. 1959). The results of this study might be of even wider interest, since, most surprisingly, to our knowledge the effects of these levels of hypothermia on the volume homeostasis of mammalian cells in general have not yet been systematically studied.