Department of Anesthesiology and Critical Care Medicine, Hokkaido University Graduate School of Medicine, N15 W7, Kita-ku, Sapporo, 060-8638 Japan*Corresponding author
Accepted for publication: April 9, 2002
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Abstract |
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Methods. Apoptosis was induced by depriving the cell culture medium of serum, which is one of the most representative methods to induce apoptosis, but not necrosis, in PC12 cells. Effects of mild (35 and 33°C) and moderate (31 and 29°C) hypothermia on apoptosis were evaluated. Cytotoxicity (lactate dehydogenase leakage) and the percentage of apoptotic cells (calculated by flow cytometry with propidium iodide) were evaluated 4 days after induction of apoptosis. As a control, cells without induction of apoptosis were incubated under the same conditions as the apoptosis group.
Results. Without induction at 37°C, cytotoxicity and the percentage of apoptotic cells were over 60 and 90%, respectively. At each temperature examined below 35°C, significant decreases in cytotoxicity and the percentage of apoptotic cells were observed. Mean cytotoxicity at 31 and 29°C was 50.2 (SD 4.2)% and 47.9 (4.4)%, respectively. The percentage of apoptotic cells at 31 and 29°C was 42.5 (7.4)% and 36.5 (7.3)%, respectively. In the control group, cytotoxicity and the percentage of apoptotic cells were significantly higher at 29°C than at 37°C.
Conclusions. Mild and moderate hypothermia (2935°C) inhibited apoptosis, although hypothermia below 30°C may induce apoptosis in intact cells.
Br J Anaesth 2002; 89: 3015
Keywords: cells, apoptosis; brain, cerebral ischaemia; hypothermia
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Introduction |
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The mechanism of cerebral protection by mild hypothermia is still unclear. However, it is not wholly attributable to metabolic inhibition, because a temperature reduction of 12°C decreases the cerebral metabolic rate of oxygen consumption by only 714%.7 In fact, Busto and colleagues also reported that cerebral energy metabolites such as ATP were depleted to a similar degree at 33, 34 and 37°C at the end of 20 min of four-vessel occlusion.3
Recently, it was reported that apoptosis was detected following focal cerebral ischaemia, especially in the penumbral region.8 9 It has also been reported that delayed neuronal death following global ischaemia may be partly attributable to apoptosis.10 11 Accordingly, it is speculated that ischaemic cell death is partly due to apoptosis in addition to necrosis.
Necrosis occurs due to intracellular energy depletion, after which metabolic depression may inhibit necrosis. In contrast, apoptosis occurs due to activation of intracellular cascades.12 Therefore, mild hypothermia may protect against cerebral ischaemia by inhibiting the intracellular apoptotic cascades activated by ischaemia. In this study, therefore, we used serum-deprived PC12 cells as the neuronal apoptotic model and examined the direct effects of mild and moderate hypothermia on apoptosis.
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Methods |
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Effects of hypothermia: cytotoxicity assay
Cells (1x105) were subcultured to 35-mm collagen-coated dishes. Two days after normal culture at 37°C, apoptosis was induced in phenol-red-free DMEM (Gibco) without serum. The cells were randomly incubated in chambers, in which the temperature was maintained at 37, 35 or 33°C (mild hypothermia series), or 37, 31 or 29°C (moderate hypothermia series), in a 100% humidified atmosphere containing 5% carbon dioxide/95% air. The temperature of each chamber was accurately maintained by a thermostat (Digimulti D611; Techno Seven, Yokohama, Japan). As a control, cells in which apoptosis was not induced were incubated under the same conditions.
Cytotoxicity was evaluated 4 days after serum deprivation using the lactate dehydrogenase (LDH) leakage assay as reported previously.14 Briefly, the culture supernatants were collected after the cells were sedimented by centrifugation. Next, they were incubated with a LDH reaction mixture using a cytotoxicity detection kit (Boehringer Mannheim, GmbH, Mannheim, Germany). Changes in absorbance at 490 nm were measured with a spectrophotometric microplate reader (model 3350; Bio-Rad, Hercules, CA, USA). LDH activity of each sample was obtained from the change in absorbance of the LDH standard. LDH activity of the cells was also measured after cells were lysed in 1% Triton X-100. Cytotoxicity (%) was defined as follows: (LDH activity that leaked from the cells)/(LDH activity that leaked from the cells + LDH activity within the cells).
Effects of hypothermia: percentage of apoptotic cells
Cells (1x105) were subcultured to 35-mm collagen-coated dishes. Two days after normal culture at 37°C, the medium was deprived of serum. The cells were randomly allocated into a mild or moderate hypothermia series as in the cytotoxicity assay. As a control, non-induced cells were incubated under the same conditions.
The method for flow cytometric analysis was as described previously.14 Briefly, cells were fixed in 70% ethanol following harvest. Fixed cells were incubated with 0.4 mg ml1 RNase A (Sigma, St Louis, MO, USA) and stained with propidium iodide (Sigma). Cellular DNA content was measured by flow cytometry (FACS CaliburTM; Becton Dickinson, San Jose, CA, USA). Calculation of the percentage of apoptotic cells was based on the cumulative frequency curves of the appropriate DNA histograms. Apoptotic cells were regarded as the population that contained less DNA than at the G1 peak (Fig. 1).
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Results |
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Percentage of apoptotic cells: apoptosis group (Fig. 3)
The percentage of apoptotic cells at 37°C was >90%. At each temperature below 35°C, this was significantly decreased compared with 37°C (P<0.01) (Fig. 3). At 29°C, this value decreased to 36.5 (7.3)%.
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Discussion |
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Studies of cytotoxicity and percentage of apoptotic cells revealed that hypothermia directly inhibited neuronal apoptosis in the mild and moderate range (2935°C). As we reported previously,14 several mechanisms such as production of ROS and induction of caspase activity are suggested as intracellular cascades leading to apoptosis induced by serum deprivation in PC12 cells. They are also observed after cerebral ischaemia.2224 Kil and colleagues examined the levels of the salicylate hydroxylation product as an index of ROS production in the rat forebrain ischaemia model at brain temperatures of 30, 36 and 39°C.17 Levels of the salicylate hydroxylation product significantly decreased in a temperature-dependent fashion. It was reported that expression of caspase-3 proteins was decreased during hypothermia at 33°C after rat forebrain ischaemia.19 Thus, the inhibition of apoptosis under hypothermia observed in this study might also be related to depression of such intracellular cascades.
Lucas and colleagues reported that there were no morphological or electrophysiological changes in spinal cord neurone culture, even when the temperature was decreased to 17°C for 2 h.25 In our study, however, cytotoxicity and the percentage of apoptotic cells significantly increased at 29°C in the control group. This result suggests that hypothermia may damage intact cells through induction of apoptosis if the period of hypothermia is lengthy, even if the range of hypothermia is moderate. The result may be noteworthy because as of late, hypothermic therapy is usually performed for several days.
In conclusion, we evaluated the relationship between hypothermia and neuronal apoptotic processes using PC12 cells, in which apoptosis but not necrosis was induced by serum deprivation. Mild and moderate hypothermia (2935°C) inhibited apoptosis, although hypothermia below 30°C may induce apoptosis.
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References |
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