H4 human neuroglioma cells (H4 cells) were used in the studies. The cells were cultured in DMEM (high glucose) containing 9% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 ug/ml streptomycin, and 2 mM L-glutamine.

Isoflurane was delivered from an anesthesia machine to a sealed plastic box in a 37°C incubator containing 6-well plates; the 6-well plates were seeded with one million cells in 1.5 ml cell culture media per well, as described in our previous studies (Zhang et al., 2012). A Dash 400 gas analyzer (General Electric Company, Boston, MA) was used to continuously monitor the delivered concentrations of carbon dioxide, oxygen, and isoflurane. The cells were treated with 2% isoflurane, plus 21% O₂, and 5% CO₂ at for a duration of 3 or 6 h as described by our previous studies (Xie et al., 2006, 2007; Ni et al., 2016). Mild hypothermia (35°C) was created by putting the sealed plastic box in a 35°C incubator.

Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, St. Louis, MO). Experiments were performed as described in the protocol provided by the company. Briefly, we added 150 μL of MTT (5 mg/mL) solution to each well, containing 1.5 mL of cell culture medium, on a 6-well plate for treatment with 2% isoflurane for 6 h. We then returned the cells to the incubator for 2 h. Finally, we removed the cell culture medium and added 1.5 mL isopropanol into each well. We spectrophotometrically measured the absorbance at a wavelength of 570 nm. We presented the changes in absorbance, as the levels of cell viability, in the cells treated with isoflurane and/or mild hypothermia as the percentage of those in the cells treated with control conditions. The reduction in cell viability (e.g., decrease in MTT levels) indicates cytotoxicity.

Cell membrane integrity or cell viability was also assessed using a commercial lactate dehydrogenase (LDH) kit (Roche Applied Science, Madison, WI). Experiments were performed as described in the protocol provided by the company. Levels of LDH released into the medium were used to reflect cell membrane integrity or cell viability. Briefly, after the treatments of isoflurane and/or mild hypothermia, we collected the cell culture medium and assessed LDH levels according to the manufacturer's instructions. We spectrophotometrically measured the absorbance at a wavelength of 490 nm. The ratio of LDH levels in the supernatant to the total LDH levels served as the index of cell viability. We presented the changes in absorbance, as the degree of cell viability, in the cells treated with isoflurane and/or mild hypothermia as the percentage of those in the cells treated with control condition. The reduction in cell membrane integrity (e.g., increase in LDH levels) or cell viability indicates cytotoxicity.

The cells were harvested at the end of the experiments for Western blot analysis. The pellets of the harvested cells were detergent-extracted on ice using an immunoprecipitation buffer plus protease inhibitors, as described in our previous studies (Zhang et al., 2012). The lysates were collected, centrifuged at 13,000 rpm for 15 min, and quantified for total protein amount by a bicinchoninic acid protein assay kit (Pierce, Rockford, IL).

Western blot analyses were performed as described in our previous studies (Zhang et al., 2010, 2012). Specifically, γH2A.X antibody (1:1000 dilution; Cell Signaling Technology, Danvers, MA) was used to detect the levels of γH2A.X (15 kDa), and β-actin antibody (1:10,000, Sigma, St. Louis, MO) was used to detect β-actin (42 kDa). Each band in the Western blot represented an independent experiment. The results were averaged from six independent experiments. The intensity of signals was analyzed using the National Institute of Health image program. We quantified the Western blots in two steps. First, we used β-actin levels to normalize protein levels (e.g., determining the ratio of γH2A.X to β-actin amount) and to control for loading differences in the total protein amount. Secondly, we presented protein level changes in cells exposed to isoflurane and/or mild hypothermia as the percentage of those in the control group. One hundred percentage of the protein level changes refer to the control levels, for the purpose of comparison to the experimental conditions.

We employed the ATP Determination Kit (Invitrogen) in the experiments to detect ATP levels, as described in our previous studies (Zhang et al., 2010, 2012). In short, the cells were placed in 6-well plates in the incubator overnight. The cells were then exposed to the isoflurane and/or mild hypothermia treatment. At the end of the treatment, the amount of fluorescence was measured, and the levels of ATP in the experimental samples were calculated from the standard curve made from samples containing known amounts of ATP.

Data were expressed as means ± standard deviation (SD). The number of samples was 6–8 per group, and the power calculation was performed using information collected from a preliminary study that was conducted under the same conditions. Based on the preliminary data, assuming a two-sided Student-t-test, samples of 6 for each control and treatment group would lead to 90% power and 95% significance. Student's t-test and one-way ANOVA were used to determine the difference between the treatment (isoflurane vs. isoflurane plus hypothermia) and control condition. Post hoc analyses (Bonferroni test) were conducted if the main effects were found to be statistically significant. P-values <0.05 were considered statistically significant. Prism 6 software (GraphPad software, La Jolla, CA) was used to analyze the data.

The objective of the current study was to find the potential methods to protect the cytotoxicity induced by anesthetic isoflurane and to illustrate the underlying mechanisms. Our previous studies had shown that treatment with 2% isoflurane for 6 h could induce cytotoxicity in vitro (Xie et al., 2006, 2007; Zhang et al., 2010, 2012). Therefore, we first assessed the effects of mild hypothermia on the isoflurane-induced cytotoxicity (e.g., cell death). The cell viability was determined by MTT assay. As can be seen in Figure 1A, MTT assay showed that treatment with 2% isoflurane for 6 h decreased the MTT levels as compared to control condition: 69.5 vs. 100%. However, mild hypothermia attenuated the isoflurane-induced reduction in MTT levels: 83.5 vs. 69.5%. One-way ANOVA showed the significant difference among these three conditions: control, isoflurane, and isoflurane plus mild hypothermia (Figure 1A, F = 22.99, ^**P = 0.001). Consistently, the mild hypothermia ameliorated the isoflurane-induced increase in LDH release: 100% (control condition) vs. 163 (isoflurane) vs. 110% (isoflurane plus mild hypothermia; Figure 1B, F = 14.42, ^**P = 0.001). These data suggest that isoflurane was able to induce the cytotoxicity in H4 cells, and mild hypothermia could attenuate the isoflurane-induced cytotoxicity in the cells.

Given the findings that mild hypothermia was able to attenuate the isoflurane-induced cytotoxicity, next we assessed whether the mild hypothermia could also attenuate the isoflurane-induced increase in the γH2A.X level, the marker of DNA damage, in the cells. The immunoblotting of γH2A.X showed that the treatment with 2% isoflurane for 6 h (lanes 5–8) induced visible increases in the density of bands representing γH2A.X as compared to control condition (lanes 1–4) (Figure 2A). There was no significant difference in β-actin level between the isoflurane treatment and control condition (Figure 2A). The quantification of the Western blot (Figure 2B), based on the ratio of γH2A.X to β-actin, showed that the isoflurane treatment (black bar) increased γH2A.X level as compared to the control condition (white bar): 348 vs. 100%, ^**P = 0.001. Next, we found that the mild hypothermia (lanes 7–9) could attenuate the isoflurane-induced increase in the γH2A.X level (lanes 4–6) (Figure 2C). The quantification of Western blot showed there was a significant difference on the levels of γH2A.X among the control condition (100%, white bar), isoflurane treatment (318%, black bar), and hypothermia plus isoflurane (108%, shadow bar; F = 18.10, ^**P = 0.0029; Figure 2D). γH2A.X is a maker of DNA damage (Bonner et al., 2008; Garcia-Canton et al., 2012; Valdiglesias et al., 2013; Ni et al., 2016), thus, these data suggest that hypothermia was able to attenuate the isoflurane-induced DNA damage.

Isoflurane has been shown to induce the cytotoxicity by decreasing ATP levels (Zhang et al., 2010, 2012). We therefore determined whether the mild hypothermia could affect the isoflurane-induced changes in ATP levels. In this experiment, we treated the cells with 3% isoflurane for 3 h, but not 6 h to test whether the short time (3 h) isoflurane treatment was able to decrease the ATP levels without causing cytotoxicity in the cells. We found that the treatment with 2% isoflurane for 3 h decreased ATP levels as compared to the control condition, and the mild hypothermia rescued the isoflurane-induced reduction in the ATP levels: control (100%), isoflurane (56%), isoflurane plus mild hypothermia (78%) (Figure 3A, F = 21.61, ^**P = 0.0001, one-way ANOVA). Moreover, we found the treatment with 2% isoflurane for 3 h did not induce cytotoxicity (MTT assay) (Figure 3B). These data showed that isoflurane-induced reduction in ATP levels might occur before the isoflurane-induced cytotoxicity. These results suggest that isoflurane causes DNA damage and induces cytotoxicity by decreasing ATP levels, which can be rescued by the mild hypothermia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JC and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.