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Meeting Abstracts  |   February 2008
The Common Inhalational Anesthetic Isoflurane Induces Apoptosis via  Activation of Inositol 1,4,5-Trisphosphate Receptors
Author Affiliations & Notes
  • Huafeng Wei, M.D., Ph.D.
    *
  • Ge Liang, M.D.
  • Hui Yang, M.D.
  • Qiujun Wang, M.D.
    §
  • Brian Hawkins, Ph.D.
  • Muniswamy Madesh, Ph.D.
    #
  • Shouping Wang, M.D.
    **
  • Roderic G. Eckenhoff, M.D.
    ††
  • * Assistant Professor, † Research Specialist, †† Professor, Department of Anesthesiology and Critical Care, ∥ Visiting Scholar/Postdoctoral Research Fellow, # Assistant Professor, Department of Cancer Biology and Institute of Environmental Medicine, University of Pennsylvania. ‡ Visiting Scholar/Postdoctoral Research Fellow, Department of Anesthesiology and Critical Care, University of Pennsylvania; Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. § Visiting Scholar/Postdoctoral Research Fellow, Department of Anesthesiology and Critical Care, University of Pennsylvania; Department of Anesthesiology, The Third Clinical Hospital, Hebei Medical University, Shijiazhuang, China. ** Visiting Scholar/Postdoctoral Research Fellow, Department of Anesthesiology and Critical Care, University of Pennsylvania; Department of Anesthesia, Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.
Article Information
Meeting Abstracts   |   February 2008
The Common Inhalational Anesthetic Isoflurane Induces Apoptosis via  Activation of Inositol 1,4,5-Trisphosphate Receptors
Anesthesiology 2 2008, Vol.108, 251-260. doi:10.1097/01.anes.0000299435.59242.0e
Anesthesiology 2 2008, Vol.108, 251-260. doi:10.1097/01.anes.0000299435.59242.0e
THE common inhalational anesthetic isoflurane induces cytotoxicity in both a concentration- and time-dependent manner in different types of cultured cells.1–8 Further, at clinically relevant concentrations, isoflurane caused widespread neuronal apoptosis in developing rat brains with subsequent persistent learning deficits.9,10 Isoflurane also caused cognitive dysfunction persisting for several weeks after treatment in adult and aged rats11,12 and aged mice.13 Therefore, it is possible that general anesthesia may contribute to the recently reported cognitive deficits after surgery, especially in aged patients.14,15 
The inositol 1,4,5-trisphosphate (IP3) receptors, found in the endoplasmic reticular (ER) membrane, play an important role in both normal physiology16 and pathologic neurodegeneration.17,18 Excessive calcium release from the ER, via  overactivation of IP3receptors, results in elevation of cytosolic calcium concentration ([Ca2+]c), calcium overload in mitochondria, and depletion of ER calcium, all of which can contribute to cell death.19,20 In addition, cytochrome c  released from mitochondria due to calcium overload removes the negative feedback inhibition of IP3receptors by cytosolic calcium, leading to a vicious cycle of excessive calcium release from the ER via  IP3receptors.17,20 Cytochrome c  release also activates caspase 3, which in turn cleaves IP3receptors, resulting in a permanent leak of calcium from the ER.21 Predictably, cells deficient in IP3receptors demonstrate resistance to apoptosis induced by a variety of injuries.22 Over activation of IP3receptors is implicated in the neurodegeneration seen in diseases such as Alzheimer disease and Huntington disease (HD) and in conditions such as stroke.18,19,23,24 
The mechanisms of isoflurane neurotoxicity are unknown. Our previous work has demonstrated that dantrolene, an ER ryanodine receptor antagonist, inhibited isoflurane-induced apoptosis,1 suggesting a role for abnormal calcium release from the ER in isoflurane neurotoxicity. In neurons, isoflurane may induce calcium release from the ER, although it is not clear whether this is the result of actions on the IP3or the ryanodine receptor.25 We hypothesized in the current study that isoflurane induces apoptosis by excessive calcium release from ER via  overactivation of IP3receptor. To test this hypothesis, we studied apoptosis and calcium release from the ER in a variety of cell types with varying IP3receptor expression or activity.
Materials and Methods
Cell Cultures
Cells of wild-type (WT) chicken B lymphocyte (DT40) cell line, and its total IP3receptor knock-out (DT40 IP3R TKO) type, were cultured in RPMI 1640 with 10% fetal calf serum, 1% chicken serum, 50 μm 2-mercaptoethanol, 4 mm l-glutamine, and antibiotics in a 95% air, 5% CO2humidified atmosphere at 38°C as previously described.26 Rat pheochromocytoma (PC12) cells transfected with WT presenilin 1 (PS1) or point mutated PS1 (L286V) were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated horse serum, 5% fetal calf serum, 200 μg/ml G418, and antibiotics in a 95% air, 5% CO2humidified atmosphere at 37°C as described.1,27 The transfection of the WT and mutant PS1 has been described and confirmed in detail previously.28,29 Mouse HD knock-in striatal cells (STHdh  Q111/Q111) and their WT control cells (STHdh  Q7/Q7) were generated and cultured as described previously.30,31 Briefly, cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 400 μg/ml G418, and antibiotics in a 95% air, 5% CO2humidified atmosphere at 33°C.
Anesthetic Exposure
DT40 cells (WT and IP3R TKO), rat PC12 cells (WT and L286V), and mouse striatal cells (WT and HD) were exposed to isoflurane at different concentrations for various durations in a gastight chamber inside the culture incubator (Bellco Glass, Inc., Vineland, NJ), with humidified 5% CO2–21% O2–balanced N2(AirGas East, Bellmawr, NJ) going through a calibrated agent-specific vaporizer as described previously.1 Gas phase concentrations in the gas chamber were verified and maintained at the desired concentration throughout the experiments using an infrared Ohmeda 5330 agent monitor (Coast to Coast Medical, Fall River, MA). In a pilot study, the cell medium was aspirated and extracted into hexane for high-performance liquid chromatography measurement (System Gold; Beckman Coulter, Fullerton, CA) to verify that the various anesthetic concentrations in the medium in millimolars are equivalent to the minimal alveolar concentration (MAC) in the gas phase inside the gas chamber using the concentration correlation previously described.32 
Imaging Analysis of Annexin V and Propidium Iodide
Translocation of membrane phospholipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane is an early indication of cell damage. Annexin V, a phospholipid binding protein with a high affinity for phospholipid phosphatidylserine, can bind to phospholipid phosphatidylserine once it is exposed to the extracellular environment. Propidium iodide (PI) can bind to nucleic acid after penetrating a breached plasma membrane, as occurs in the later stages of cell damage. We treated DT40 cells, grown floating in the medium, with different concentrations of isoflurane (0.6, 1.2, and 2.4%) for 24 h, as well as with 2.4% isoflurane for different times (6, 12, and 24 h). Immediately after treatment, we determined annexin V– or PI–positive cells by the methods described previously.26 Cells were dropped onto 25-mm cover slips and stained with annexin V or PI. The stained cells were visualized and counted by two persons blinded to the treatments. The percentage of annexin V– or PI–positive cells averaged from four areas on each cover slip was then calculated and compared.
Detection of Caspase-3 Activity
Increased caspase-3 activity is a typical marker for apoptosis. The assay is based on the ability of the active enzymes to cleave the fluorogenic substrates Ac-DEVD-AFC (caspase 3; Calbiochem, San Diego, CA) and was performed as per instructions and as described previously.26 DT40 cells grown on six well plates were treated with 2.4% isoflurane for 24 h and then were harvested via  trypsinization and washed with phosphate-buffered saline. The cell pellet was gently resuspended in CelLytic M lysis buffer and protease inhibitor cocktail (Sigma, St. Louis, MO), lysed, and centrifuged; the supernatant was used for the assay. Caspase substrates were added to a final concentration of 50 μm, and the samples were incubated at 37°C for 45 min in caspase assay buffer. Incubated samples were measured at an excitation of 400 nm and an emission of 505 nm in a multiwavelength-excitation dual wavelength-emission fluorometer (Delta RAM; photon Technology International, Birmingham, NJ). We determined the caspase-3 activity immediately after treating DT40 cells with 2.4% isoflurane for 24 h.
Cytotoxicity Assays
The inhibition of isoflurane-induced cytotoxicity by xestospongin C, a potent antagonist of IP3receptor, was assessed by the lactate dehydrogenase (LDH) release assay in PC12 cells and MTS (3-(4,5-dimethylthiazol-2-yl)- 5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) reduction assay in mouse striatal cells. LDH release reflects plasma membrane integrity, is a relatively late event in cytotoxicity, and is shared by both apoptosis and necrosis pathways, and its measurement is well described.1 The MTS reduction assay reflects mitochondrial function and is considered a middle event in both apoptosis and necrosis. The tetrazolium compound is bioreduced by normal mitochondria into a colored formazan product measured by absorbance. We followed the standard experimental protocol for MTS reduction assay from Promega (Madison, WI). We treated PC12 and striatal neurons, grown on 24 well plates, with 2.4% isoflurane for 24 h. Immediately (PC12 cells) or 24 h (striatal neurons) after treatment, LDH or MTS assay was performed, respectively. Xestospongin C at 100 nm was used to inhibit IP3receptor activity. The results of LDH release and MTS reduction assays were expressed as a percentage of the control without anesthetic treatment.
Simultaneous Confocal Imaging of Cytosolic and Mitochondrial Ca2+
The method used was the same as described previously.26 DT40 cells (WT and IP3R TKO) grown on 25-mm cover slips were loaded with 2 μm rhod-2/AM in cell medium containing 2.0% bovine serum albumin in the presence of 0.003% Pluronic acid at 37°C for 50 min. Cells loaded with rhod-2 dye were washed and then reloaded with fluo-4/AM for an additional 30 min at room temperature. Cells were placed on a stage and exposed to 2 MAC (0.7 mm) isoflurane dissolved in the perfusion buffer. The images were recorded using the Radiance 200 imaging system with excitation at 488 and 568 nm for fluo-4 and rhod-2, respectively.
Measurement of Cytosolic Calcium Concentration
Cytosolic calcium concentration was measured using fura-2 fluorescence (Molecular Probes, Eugene, OR) with a photometer coupled to an Olympus 1Χ70 inverted microscope (Olympus America Inc., Center Valley, PA) and IPLab version 3.7 imaging processing and analysis software (Biovision Technologies, Exton, PA). The protocol to determine [Ca2+]cwas similar to that previously described, with some modifications.33 Briefly, cells grown on 25-mm round glass cover slips were washed three times with Krebs-Ringer’s buffer without addition of calcium and then loaded with 2.5 μm fura-2/AM (Molecular Probes) for 30 min at room temperature. The cells were then placed in a sealed chamber (Warner Instrument Inc., Hamden, CT) connected with multiple inflow infusion tubes and one outflow tube, which provided constant flow to the chamber. The cells were first washed with Krebs-Ringer’s buffer through one inflow tube for the baseline measurement of [Ca2+]c, and then were exposed to isoflurane via  a separate inflow infusion tubes driven by a syringe pump (Braintree Scientific Inc., Braintree, MA). The fluorescence signals were measured with excitation at 340 and 380 alternatively and emission at 510 nm for a period up to 18 min for each treatment. A pilot study confirmed that the cells were still viable at the end of experiments for calcium measurement. The fluorescence measurements were calibrated by bathing cells in the HEPES buffer containing ionomycin 20 mm for maximum or 20 mm EGTA for minimum calcium values. The intracellular calcium concentration ([Ca2+]i) was calculated by the ratio method of Grynkiewicz et al.  ,34 using 224 nm as the Kd of fura-2. The final result of [Ca2+]cwas averaged from the cells of at least three separate experiments. We used 0.7 mm (2 MAC) isoflurane to elevate [Ca2+]cin all cells. Xestospongin C at 1 μm was used to inhibit isoflurane-mediated calcium release from the ER.
Statistics
We used GraphPad Prism 4 software (GraphPad Software, Inc., San Diego, CA) and STATA version 7 Software (StataCorp LP, College Station, TX) for all statistical analysis. Annexin V and PI staining, LDH release, and MTS reduction were all expressed as percentage of those in the control. Peak [Ca2+]cin PC12 and HD striatum cells was expressed as a percentage of its own baseline. We analyzed the data with one-way analysis of variance followed by Newman-Keuls multiple comparison tests using GraphPad Prism. We also confirm our analysis of dose response and time response of image analysis of cell damage marker annexin V and PI in DT40 cells with a mixed mode of regression using STATA version 7. A significantly increased odds ratio risk was determined if the estimate for the greater time point or dose exceeded the upper limit of the 95% confidence interval for the lower time point or dose. P  < 0.05 was considered statistically significant.
Results
DT40 IP3R TKO Cells Were Resistant to Isoflurane-induced Apoptosis
To investigate whether isoflurane induces apoptosis in the absence of IP3receptors, we first studied its cytotoxic effects in the DT40 IP3R TKO cells and compared it with the corresponding WT cells. Isoflurane induced cell damage determined by both annexin V and PI staining concentration-dependently in WT but not in IP3R TKO cells (figs. 1A–C). The lowest concentration of isoflurane required to induce apoptosis in WT cells was 1.2% (figs. 1B and C), a clinically used concentration. Isoflurane also induced cell damage time-dependently in WT but not TKO cells (figs. 1D–F). In WT cells, 2.4% isoflurane induced early cell damage in as little as 6 h of treatment (fig. 1E), a duration often used for surgeries. To confirm that isoflurane induced cell damage in DT40 cells by apoptosis, we measured changes of caspase-3 activity after exposing these cells to 2.4% isoflurane for 24 h. Isoflurane caused a dramatic elevation (more than fourfold) of caspase-3 activity in DT40 WT but not IP3R TKO cells (fig. 2A).
Fig. 1. Inositol 1,4,5-trisphosphate (IP3) receptor total knock-out chicken lymphocytes display resistance to isoflurane-induced cell damage. The early cell damage was determined by the externalization of anionic phospholipid, phosphatidyl serine–annexin V binding (annexin V). The late cell damage was determined by propidium iodide (PI) staining, representing loss of plasma membrane integrity. Isoflurane induced cell damage dose-dependently (  A    C  ) and time-dependently (  D    F  ) in DT40 wild-type (WT) but not IP3receptor total knock-out cells. Representative imaging of annexin V–positive cells (  green  ) and PI-positive cells (  red  ) after treatment of isoflurane at various concentrations for 24 h (  A  ) or 2.4% isoflurane for various times (  D  ) in WT or IP3receptor total knock-out cells.  Scale bar  = 50 μm. Isoflurane for 24 h induced early (  B  ) and late (  C  ) cell damage dose-dependently in WT but not IP3receptor total knock-out cells. Isoflurane at 2.4% induced early (  E  ) and late (  F  ) cell damage time-dependently in WT but not IP3receptor total knock-out cells. Data represent mean ± SE from nine repeats of three separate experiments. ** or ***  P  < 0.01 or  P  < 0.001 compared with its corresponding control in WT cells. ###  P  < 0.001 compared with its own baseline before the isoflurane treatment. ISO = isoflurane; TKO = IP3receptor total knock-out. 
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Fig. 1. Inositol 1,4,5-trisphosphate (IP3) receptor total knock-out chicken lymphocytes display resistance to isoflurane-induced cell damage. The early cell damage was determined by the externalization of anionic phospholipid, phosphatidyl serine–annexin V binding (annexin V). The late cell damage was determined by propidium iodide (PI) staining, representing loss of plasma membrane integrity. Isoflurane induced cell damage dose-dependently (  A    C  ) and time-dependently (  D    F  ) in DT40 wild-type (WT) but not IP3receptor total knock-out cells. Representative imaging of annexin V–positive cells (  green  ) and PI-positive cells (  red  ) after treatment of isoflurane at various concentrations for 24 h (  A  ) or 2.4% isoflurane for various times (  D  ) in WT or IP3receptor total knock-out cells.  Scale bar  = 50 μm. Isoflurane for 24 h induced early (  B  ) and late (  C  ) cell damage dose-dependently in WT but not IP3receptor total knock-out cells. Isoflurane at 2.4% induced early (  E  ) and late (  F  ) cell damage time-dependently in WT but not IP3receptor total knock-out cells. Data represent mean ± SE from nine repeats of three separate experiments. ** or ***  P  < 0.01 or  P  < 0.001 compared with its corresponding control in WT cells. ###  P  < 0.001 compared with its own baseline before the isoflurane treatment. ISO = isoflurane; TKO = IP3receptor total knock-out. 
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Fig. 2. Inositol 1,4,5-trisphosphate (IP3) receptor total knock-out chicken lymphocytes were resistant to isoflurane-induced apoptosis and calcium elevations. (  A  ) Isoflurane at 2.4% for 24 h dramatically increased caspase-3 activity in wild-type (WT) but not IP3receptor total knock-out chicken lymphocytes. Data represent mean ± SE from three experiments. ***  P  < 0.001 compared with control. (  B  ) Isoflurane at 2.4% for 24 h significantly increased propidium iodide–positive cells (late cell damage) only in WT but not IP3receptor total knock-out cells, whereas thapsigargin (inhibitor of calcium adenosine triphosphatase on endoplasmic reticulum membrane as a positive control) still significantly increased propidium iodide–positive cells in both WT and IP3receptor total knock-out cells (n = 9; ***  P  < 0.001 compared with control). (  C  ) Representative tracing reveals rapid Ca2+mobilization followed by mitochondrial Ca2+uptake, as indicated by an increase in fluo-4 (  green  ) fluorescence and a subsequent increase in rhod-2 fluorescence in the mitochondria (  red  ) after exposure to 0.7 mm (2 minimum alveolar concentration) isoflurane or 1 μm thapsigargin. The same results were repeated in three separate experiments. Isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) precedes increase of [Ca2+]monly in WT (  C  ) but not in IP3receptor total knock-out cells (  D  ). Thapsigargin still significantly elevated both [Ca2+]cand mitochondria calcium concentration ([Ca2+]m) in IP3receptor total knock-out cells (  E  ). DT40 = chicken B lymphocytes; ISO = isoflurane; Tg = thapsigargin; TKO = IP3receptor total knock-out chicken B lymphocytes. 
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Fig. 2. Inositol 1,4,5-trisphosphate (IP3) receptor total knock-out chicken lymphocytes were resistant to isoflurane-induced apoptosis and calcium elevations. (  A  ) Isoflurane at 2.4% for 24 h dramatically increased caspase-3 activity in wild-type (WT) but not IP3receptor total knock-out chicken lymphocytes. Data represent mean ± SE from three experiments. ***  P  < 0.001 compared with control. (  B  ) Isoflurane at 2.4% for 24 h significantly increased propidium iodide–positive cells (late cell damage) only in WT but not IP3receptor total knock-out cells, whereas thapsigargin (inhibitor of calcium adenosine triphosphatase on endoplasmic reticulum membrane as a positive control) still significantly increased propidium iodide–positive cells in both WT and IP3receptor total knock-out cells (n = 9; ***  P  < 0.001 compared with control). (  C  ) Representative tracing reveals rapid Ca2+mobilization followed by mitochondrial Ca2+uptake, as indicated by an increase in fluo-4 (  green  ) fluorescence and a subsequent increase in rhod-2 fluorescence in the mitochondria (  red  ) after exposure to 0.7 mm (2 minimum alveolar concentration) isoflurane or 1 μm thapsigargin. The same results were repeated in three separate experiments. Isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) precedes increase of [Ca2+]monly in WT (  C  ) but not in IP3receptor total knock-out cells (  D  ). Thapsigargin still significantly elevated both [Ca2+]cand mitochondria calcium concentration ([Ca2+]m) in IP3receptor total knock-out cells (  E  ). DT40 = chicken B lymphocytes; ISO = isoflurane; Tg = thapsigargin; TKO = IP3receptor total knock-out chicken B lymphocytes. 
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Isoflurane-induced Apoptosis Was Associated with Elevation of [Ca2+]cand Then [Ca2+]min DT40 WT but Not IP3R TKO Cells
To further examine the hypothesis that isoflurane induced apoptosis by disruption of intracellular calcium homeostasis, we first assayed the cytotoxic effects of thapsigargin, a selective inhibitor of the ER calcium adenosine triphosphatase, which can cause ER calcium to passively leak even without any contribution from IP3receptors.35 Thus, in this positive control experiment, thapsigargin (100 nm for 2 h) induced similar apoptosis in both WT and TKO cells (fig. 2B), whereas 2.4% isoflurane for 24 h only induced apoptosis in DT40 WT cells. Isoflurane induced a sequential elevation of [Ca2+]cand then mitochondria calcium concentration ([Ca2+]m) only in DT40 WT but not TKO cells (figs. 2C and D), whereas thapsigargin produced this sequential calcium elevation in both WT and IP3R TKO cells (fig. 2E). These results show that the IP3receptor knock-out did not diminish the cells response to stress or calcium transients in general, and thereby strengthen the hypothesis that isoflurane induced apoptosis by causing calcium release from the ER via  overactivation of IP3receptors.
Elevated Activity of IP3Receptors Enhances Isoflurane-induced Apoptosis
The mutation of PS1, a protein located primarily on ER membranes, appears in most cases of familial Alzheimer disease, and is associated with increased expression of ryanodine receptors27 and increased activity of IP3receptors.36,37 If isoflurane induces apoptosis by overactivation of IP3receptor, cells with enhanced expression or activity of this receptor should be more vulnerable to isoflurane-induced cytotoxicity and exhibit greater calcium release from the ER. Consistent with this hypothesis, isoflurane caused significantly more PI-positive cells in PC12 cells transfected with L286V PS1 than in its WT vector control (figs. 3A and B). To establish the linkage of these cytotoxic effects of isoflurane to IP3receptors, we coincubated cells with xestospongin C, a potent inhibitor of the IP3receptor.38,39 Xestospongin C abolished the cell damage represented by elevated LDH (fig. 3C) and also nearly abolished the calcium release from the ER caused by isoflurane (figs. 3D and E) in L286V PC12 cells. Although xestospongin C may also inhibit calcium adenosine triphosphatase in some cell cultures,40 it did not affect adenosine triphosphatase activity in this system because xestospongin C alone did not increase [Ca2+]cin L286V PC12 cells (data not shown).
Fig. 3. The inositol 1,4,5-trisphosphate (IP3) receptor antagonist xestospongin C (Xc) inhibited isoflurane-induced cytotoxicity and calcium elevations in PC12 cells transfected with L286V presenilin 1. (  A  ) Representative images of phase contrast (PC) or propidium iodide (PI) staining of L286V or wild-type (WT) PC12 cells immediately after treatment of 2.4% isoflurane for 24 h.  Scale bar  = 100 μm. (  B  ) Quantification of percentage of PI-positive cells (late cell damage) after treating L286V or WT PC12 cells with 2.4% isoflurane for 24 h. L286V PC12 cells were more vulnerable than WT PC12 cells to isoflurane-induced cytotoxicity. Data represent mean ± SE from 25 repeats of three separate experiments. *** or ###  P  < 0.001 compared with control in L286V or isoflurane treatment in WT, respectively. (  C  ) Pretreatment of 100 nm Xc for 30 min abolished the lactate dehydrogenase (LDH) release (late cell damage) induced by 2.4% isoflurane for 24 h in L286V cells. Data represent mean ± SE from 12 repeats of three separate experiments. **  P  < 0.01 compared with control. ##  P  < 0.01 compared with isoflurane treatment alone. (  D  ) Representative tracing reveals averaged isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) with or without pretreatment of Xc in the absence of extracellular calcium in both WT and L286V PC12 cells. (  E  ) Isoflurane at 2.4% induced significantly higher peak elevation of [Ca2+]cin the absence of extracellular calcium in L286V than in WT PC12 cells. Xc (1 μm) significantly inhibited isoflurane-induced peak elevation of [Ca2+]cin the absence of extracellular calcium in both WT and L286V cells. Data represent mean ± SE from three separate experiments. * or **  P  < 0.05 or  P  < 0.01 compared with isoflurane treatment alone without Xc pretreatment. ##  P  < 0.01 compared with WT PC12 cells treated with isoflurane alone. ISO = isoflurane; L286V = PC12 cells with presenilin-1 mutation. 
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Fig. 3. The inositol 1,4,5-trisphosphate (IP3) receptor antagonist xestospongin C (Xc) inhibited isoflurane-induced cytotoxicity and calcium elevations in PC12 cells transfected with L286V presenilin 1. (  A  ) Representative images of phase contrast (PC) or propidium iodide (PI) staining of L286V or wild-type (WT) PC12 cells immediately after treatment of 2.4% isoflurane for 24 h.  Scale bar  = 100 μm. (  B  ) Quantification of percentage of PI-positive cells (late cell damage) after treating L286V or WT PC12 cells with 2.4% isoflurane for 24 h. L286V PC12 cells were more vulnerable than WT PC12 cells to isoflurane-induced cytotoxicity. Data represent mean ± SE from 25 repeats of three separate experiments. *** or ###  P  < 0.001 compared with control in L286V or isoflurane treatment in WT, respectively. (  C  ) Pretreatment of 100 nm Xc for 30 min abolished the lactate dehydrogenase (LDH) release (late cell damage) induced by 2.4% isoflurane for 24 h in L286V cells. Data represent mean ± SE from 12 repeats of three separate experiments. **  P  < 0.01 compared with control. ##  P  < 0.01 compared with isoflurane treatment alone. (  D  ) Representative tracing reveals averaged isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) with or without pretreatment of Xc in the absence of extracellular calcium in both WT and L286V PC12 cells. (  E  ) Isoflurane at 2.4% induced significantly higher peak elevation of [Ca2+]cin the absence of extracellular calcium in L286V than in WT PC12 cells. Xc (1 μm) significantly inhibited isoflurane-induced peak elevation of [Ca2+]cin the absence of extracellular calcium in both WT and L286V cells. Data represent mean ± SE from three separate experiments. * or **  P  < 0.05 or  P  < 0.01 compared with isoflurane treatment alone without Xc pretreatment. ##  P  < 0.01 compared with WT PC12 cells treated with isoflurane alone. ISO = isoflurane; L286V = PC12 cells with presenilin-1 mutation. 
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We also examined isoflurane effects in another model of elevated IP3receptor activity. Abnormal calcium release from ER via  elevated activity of IP3receptors is thought to play an important role in the neurodegeneration of HD.23,41,42 Mutated Huntingtin protein with an enlarged polyglutamine repeat section causes excessive calcium release from the ER upon the activation of IP3receptors by an agonist.23 Therefore, if isoflurane causes apoptosis via  activation of IP3receptors, the elevated activity of IP3receptors in mouse striatal cells with an overexpression of Q111 Huntingtin should not only render these cells more vulnerable to isoflurane-induced neurotoxicity and calcium release from the ER, but also xestospongin C should inhibit these effects. In accordance with this hypothesis, 2.4% isoflurane for 24 h induced more cell damage (PI-positive cells) in Q111 Huntingtin knock-in than in the corresponding WT control striatal cells (figs. 4A and B), and xestospongin C (100 nm) abolished this cytotoxic effect (fig. 4C). In the absence of extracellular calcium, 2.4% isoflurane (0.7 mm) induced significantly greater elevation of [Ca2+]c(representing calcium release from the ER) in Q111 Huntingtin knock-in than the WT control striatal cells, an effect that was also nearly abolished by pretreatment of xestospongin C (figs. 4D and E).
Fig. 4. Xestospongin C (Xc) inhibited isoflurane-induced cytotoxicity and calcium elevations in mutated Huntington disease (HD) knocked-in striatal cells. (  A  ) Representative images of phase contrast (PC) or propidium iodide (PI) staining of HD or wild-type (WT) striatal cells at 24 h after completion of treating cells with 2.4% isoflurane for 24 h.  Scale bar  = 100 μm. (  B  ) Isoflurane at 2.4% for 24 h significantly increased percentage of cell damage determined by PI staining in HD knocked-in but not in WT striatal cells. Data represent mean ± SE of 18 repeats from three separate experiments. *  P  < 0.05 compared with control in HD striatal cells. (  C  ) Pretreatment of 100 nm Xc for 30 min abolished the MTS reduction induced by 2.4% isoflurane for 24 h in HD striatal cells. Data represent mean ± SE from 12 repeats of three separate experiments. *** or ###  P  < 0.001 compared with control or isoflurane treatment alone. (  D  ) Representative tracing reveals averaged isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) with or without pretreatment of Xc in the absence of extracellular calcium in both WT and HD cells. (  E  ) Isoflurane at 2.4% induced significantly higher peak elevation of [Ca2+]cin the absence of extracellular calcium in HD knocked-in than in WT striatal cells. Xc (1 μm) nearly abolished isoflurane-induced peak elevation of [Ca2+]cin the absence of extracellular calcium in both WT and HD striatal cells. Data represent mean ± SE from three separate experiments. **  P  < 0.01 compared with isoflurane treatment alone without Xc pretreatment. ##  P  < 0.01 compared with WT striatal cells treated with isoflurane alone. ISO = isoflurane; MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. 
Image Not Available
Fig. 4. Xestospongin C (Xc) inhibited isoflurane-induced cytotoxicity and calcium elevations in mutated Huntington disease (HD) knocked-in striatal cells. (  A  ) Representative images of phase contrast (PC) or propidium iodide (PI) staining of HD or wild-type (WT) striatal cells at 24 h after completion of treating cells with 2.4% isoflurane for 24 h.  Scale bar  = 100 μm. (  B  ) Isoflurane at 2.4% for 24 h significantly increased percentage of cell damage determined by PI staining in HD knocked-in but not in WT striatal cells. Data represent mean ± SE of 18 repeats from three separate experiments. *  P  < 0.05 compared with control in HD striatal cells. (  C  ) Pretreatment of 100 nm Xc for 30 min abolished the MTS reduction induced by 2.4% isoflurane for 24 h in HD striatal cells. Data represent mean ± SE from 12 repeats of three separate experiments. *** or ###  P  < 0.001 compared with control or isoflurane treatment alone. (  D  ) Representative tracing reveals averaged isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) with or without pretreatment of Xc in the absence of extracellular calcium in both WT and HD cells. (  E  ) Isoflurane at 2.4% induced significantly higher peak elevation of [Ca2+]cin the absence of extracellular calcium in HD knocked-in than in WT striatal cells. Xc (1 μm) nearly abolished isoflurane-induced peak elevation of [Ca2+]cin the absence of extracellular calcium in both WT and HD striatal cells. Data represent mean ± SE from three separate experiments. **  P  < 0.01 compared with isoflurane treatment alone without Xc pretreatment. ##  P  < 0.01 compared with WT striatal cells treated with isoflurane alone. ISO = isoflurane; MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. 
×
Discussion
Our results are most consistent with the hypothesis that isoflurane, a commonly used inhalational anesthetic, induces cell apoptosis by excessive calcium release from the ER via  overactivation of IP3receptors. First, cells depleted of IP3receptors were resistant to isoflurane-induced apoptosis and calcium elevation, whereas cells with enhanced activity of the IP3receptor were more vulnerable to isoflurane-induced apoptosis. Finally, the IP3receptor antagonist xestospongin C significantly inhibited isoflurane-induced cell damage and calcium elevations in the vulnerable cell models.
Our results suggest a direct interaction between isoflurane and the receptor protein, but cannot prove it. Alternative possibilities are that isoflurane interacts with an associated protein or signaling system, or the ER lipid membrane itself. But it has been demonstrated that anesthetics similar to isoflurane can bind specifically to many membrane proteins,43 so it is plausible that the effect on IP3receptors may be the result of a direct interaction.
Consistent with our previous study and others,1,44 isoflurane induced apoptosis in DT40 WT cells concentration- and time-dependently. It is important to note that there exists considerable variation in cell sensitivity to isoflurane-induced apoptosis. For example, the minimal time to induce apoptosis in DT40 cells with 2.4% isoflurane was only 6 h, with a minimal concentration of 1.2%. In rat cerebral cortical neurons or PC12 cells, the minimal concentration and time needed to induce apoptosis was 2.4% isoflurane for 24 h.1 Immortalized striatal neurons also needed a minimal exposure of 2.4% isoflurane for 24 h to induce modest cell damage in this study. In normal human peripheral lymphocytes, only 0.85% isoflurane was needed to induce apoptosis,44 and it is likely that normal human neurons are similarly sensitive. Although this study suggests that the IP3receptor is an important target underlying isoflurane-induced apoptosis, this variability in sensitivity suggests that there may be other targets that contribute, or that downstream effects are considerably different. In addition, the shortest times for 2.4% isoflurane to induce apoptosis in chicken lymphocytes are 6 h (fig. 1E) and 12 h (fig. 1F), respectively, when techniques to detect early-phase (annexin V) apoptosis or late-phase (PI staining, primarily necrosis) cell damage were used. This demonstrates that the detection of vulnerability to isoflurane cytotoxicity will vary depending on the assays used to measure cell damage. Isoflurane may produce only modestly enhanced LDH release in PS1 mutated PC12 cells (fig. 3C) because the LDH method detects severe late cell damage, whereas the cell injury induced by these concentrations of isoflurane must be more subtle, given the lack of overt clinical sequelae. Further studies are needed to determine the features that underlie this differing vulnerability to isoflurane-induced toxicity.
Increased calcium release from the ER via  the IP3receptors may contribute to neurodegeneration in other conditions, such as Alzheimer disease and HD.36,37,45 Various mutations of PS1, a protein located primarily in ER membranes, appear in most cases of familial Alzheimer disease. This PS1 mutation has been associated with increased activity of the IP3receptors,36,37 which may render neurons more vulnerable to apoptosis induced by a variety of factors that trigger calcium release (e.g.  , excitotoxicity). Our results are consistent with this hypothesis in that PC12 with L286V PS1 were more vulnerable to isoflurane cytotoxicity than its WT control, in a xestospongin C–dependent manner. It should be noted that many other PS1 mutations, such as PS1 delta 9, M146V, or just enhanced expression of full-length PS1, have been shown to contribute to cell apoptosis.46–48 If these other mutations enhance IP3receptor activity, our data would predict enhanced vulnerability to isoflurane cytotoxicity. Isoflurane may contribute to pathogenesis via  other mechanisms as well. For example, isoflurane has been recently reported to increase β-amyloid production, aggregation, and neurotoxicity2,6,8 and may be associated with tau hyperphosphorylation.49 Thus, there is a growing concern regarding the use of isoflurane in patients with symptoms of, or vulnerabilities to, neurodegenerative disease. Although the connection between polyglutamine-containing proteins and IP3receptors is not yet clear, the observations with respect to isoflurane are analogous.
It is not yet clear whether all anesthetics activate IP3receptors. Sevoflurane, a relatively new inhalational anesthetic, did not induce similar neuronal apoptosis as isoflurane at equipotent concentrations,1 so there is hope that other anesthetic drugs might prove to be less neurotoxic, at least in in vitro  studies. Clinical studies are ultimately required to demonstrate whether these cell culture studies have any relevance to humans.
It should be noted that isoflurane has been long considered an agent for cardioprotection and neuroprotection.50–52 It is likely that isoflurane is both neurotoxic and neuroprotective, depending on the concentration and duration of exposure, and the degree of patient vulnerability. Isoflurane may be inherently cytotoxic, but it provides cardioprotection or neuroprotection via  a preconditioning mechanism, much like hypoxia. Mild calcium release from the ER and moderate elevation of [Ca2+]cby isoflurane at low concentrations and short duration may trigger the ER stress response, marked by the expression of genes characterizing the well-known “preconditioning” effect.53,54 Longer exposures to isoflurane, producing extensive and prolonged calcium release from ER, may deplete ER calcium and shut down protein synthesis, leading to “cytotoxicity” effects.1,55,56 Therefore, like ischemic preconditioning, isoflurane for short durations might provide cytoprotection via  preconditioning, whereas prolonged exposures produce cytotoxicity directly. This hypothesis was supported by our recent experiments, which demonstrated that 2.4% isoflurane preconditioning for 1 h abolished neurotoxicity induced by isoflurane itself for 24 h in cerebral cortical neurons.57 These potential dual features of isoflurane should be considered in future studies, and perhaps in clinical application in vulnerable populations.
This study has several limitations that should influence in vivo  interpretations: (1) The cells with total IP3R knock-out are chicken B lymphocytes, not neurons. Although the ER calcium handling mechanisms are thought to be the same, downstream effects of the calcium transients might be considerably different. To our knowledge, there are no immortal neuronal cell lines with IP3R knock-out, but transgenic mice with type I IP3R58 or type II and III59 knock-out have been reported, from which cultures of primary neurons could be studied. Expression of IP3receptors could also be reduced via  small interfering RNA in normal neurons. (2) All of the results from this study are from cell lines, which are immortal transformed cells. Although clearly different from normal cells, in general, such cells are more resistant to stressors, making the results of our study somewhat more relevant to the in vivo  situation. Future in vivo  studies are needed to investigate whether IP3R also plays a role in isoflurane-mediated neurotoxicity in animals or patients.
Taken together, our findings suggest that the commonly used inhalational anesthetic isoflurane may induce cell apoptosis by triggering abnormal calcium release from the ER via  activation of IP3receptors. Preexisting genetic features may render some populations at increased risk from this effect.
The authors thank Mark P. Mattson, Ph.D. (Chief), and Sic L. Chan, Ph.D. (Senior Research Fellow, Laboratory of Neurosciences, National Institute on Aging, Baltimore, Maryland), for providing PC12 cells transfected with Presenilin-1 mutation; Marcy MacDonald, Ph.D. (Associate Professor, Molecular Neurogenetics Unit, Center for Human Genetic Research, Harvard University, Boston, Massachusetts), for providing striatal cells with knocked-in mutated Huntingtin protein; and Tomohiro Kurosaki, M.D., Ph.D. (Professor, Institute of Physical and Chemical Research, Research Center for Allergy and Immunology, Turumi-ku, Yokohama, Kanagawa, Japan), for providing total IP3receptor knock-out chicken lymphocytes. The authors appreciate the technical support from Qingcheng Meng, Ph.D. (Senior Research Scientist), statistical analysis by Adam Burkey, M.D. (Assistant Professor), and the editing contributions of Christopher Ward, M.D. (Anesthesia Resident, Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, Pennsylvania). The authors also appreciate the discussions with Randall Pittman, Ph.D. (Professor at the Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania).
References
Wei H, Kang B, Wei W, Liang G, Meng QC, Li Y, Eckenhoff RG: Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Res 2005; 1037:139–47Wei, H Kang, B Wei, W Liang, G Meng, QC Li, Y Eckenhoff, RG
Eckenhoff RG, Johansson JS, Wei H, Carnini A, Kang B, Wei W, Pidikiti R, Keller JM, Eckenhoff MF: Inhaled anesthetic enhancement of amyloid-beta oligomerization and cytotoxicity. Anesthesiology 2004; 101:703–9Eckenhoff, RG Johansson, JS Wei, H Carnini, A Kang, B Wei, W Pidikiti, R Keller, JM Eckenhoff, MF
Kim H, Oh E, Im H, Mun J, Yang M, Khim JY, Lee E, Lim SH, Kong MH, Lee M, Sul D: Oxidative damages in the DNA, lipids, and proteins of rats exposed to isofluranes and alcohols. Toxicology 2006; 220:169–78Kim, H Oh, E Im, H Mun, J Yang, M Khim, JY Lee, E Lim, SH Kong, MH Lee, M Sul, D
Kvolik S, Glavas-Obrovac L, Bares V, Karner I: Effects of inhalation anesthetics halothane, sevoflurane, and isoflurane on human cell lines. Life Sci 2005; 77:2369–83Kvolik, S Glavas-Obrovac, L Bares, V Karner, I
Loop T, Dovi-Akue D, Frick M, Roesslein M, Egger L, Humar M, Hoetzel A, Schmidt R, Borner C, Pahl HL, Geiger KK, Pannen BH: Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro  . Anesthesiology 2005; 102:1147–57Loop, T Dovi-Akue, D Frick, M Roesslein, M Egger, L Humar, M Hoetzel, A Schmidt, R Borner, C Pahl, HL Geiger, KK Pannen, BH
Xie Z, Dong Y, Maeda U, Alfille P, Culley DJ, Crosby G, Tanzi RE: The common inhalation anesthetic isoflurane induces apoptosis and increases amyloid beta protein levels. Anesthesiology 2006; 104:988–94Xie, Z Dong, Y Maeda, U Alfille, P Culley, DJ Crosby, G Tanzi, RE
Xie Z, Dong Y, Maeda U, Moir R, Inouye SK, Culley DJ, Crosby G, Tanzi RE: Isoflurane-induced apoptosis: A potential pathogenic link between delirium and dementia. J Gerontol A Biol Sci Med Sci 2006; 61:1300–6Xie, Z Dong, Y Maeda, U Moir, R Inouye, SK Culley, DJ Crosby, G Tanzi, RE
Xie Z, Dong Y, Maeda U, Moir RD, Xia W, Culley DJ, Crosby G, Tanzi RE: The inhalation anesthetic isoflurane induces a vicious cycle of apoptosis and amyloid beta-protein accumulation. J Neurosci 2007; 27:1247–54Xie, Z Dong, Y Maeda, U Moir, RD Xia, W Culley, DJ Crosby, G Tanzi, RE
Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF: Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003; 23:876–82Jevtovic-Todorovic, V Hartman, RE Izumi, Y Benshoff, ND Dikranian, K Zorumski, CF Olney, JW Wozniak, DF
Ma D, Williamson P, Januszewski A, Nogaro MC, Hossain M, Ong LP, Shu Y, Franks NP, Maze M: Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain. Anesthesiology 2007; 106:746–53Ma, D Williamson, P Januszewski, A Nogaro, MC Hossain, M Ong, LP Shu, Y Franks, NP Maze, M
Culley DJ, Baxter MG, Yukhananov R, Crosby G: Long-term impairment of acquisition of a spatial memory task following isoflurane–nitrous oxide anesthesia in rats. Anesthesiology 2004; 100:309–14Culley, DJ Baxter, MG Yukhananov, R Crosby, G
Culley DJ, Baxter M, Yukhananov R, Crosby G: The memory effects of general anesthesia persist for weeks in young and aged rats. Anesth Analg 2003; 96:1004–9Culley, DJ Baxter, M Yukhananov, R Crosby, G
Bianchi SL, Tran T, Liu C, Lin S, Li Y, Keller JM, Eckenhoff RG, Eckenhoff MF: Brain and behavior changes in 12-month-old Tg2576 and nontransgenic mice exposed to anesthetics [published on-line ahead of print March 6, 2007]. Neurobiol Aging
Abildstrom H, Rasmussen LS, Rentowl P, Hanning CD, Rasmussen H, Kristensen PA, Moller JT: Cognitive dysfunction 1–2 years after non-cardiac surgery in the elderly. ISPOCD group. International Study of Post-Operative Cognitive Dysfunction. Acta Anaesthesiol Scand 2000; 44:1246–51Abildstrom, H Rasmussen, LS Rentowl, P Hanning, CD Rasmussen, H Kristensen, PA Moller, JT
Moller JT, Cluitmans P, Rasmussen LS, Houx P, Rasmussen H, Canet J, Rabbitt P, Jolles J, Larsen K, Hanning CD, Langeron O, Johnson T, Lauven PM, Kristensen PA, Biedler A, van Beem H, Fraidakis O, Silverstein JH, Beneken JE, Gravenstein JS: Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post-Operative Cognitive Dysfunction. Lancet 1998; 351:857–61Moller, JT Cluitmans, P Rasmussen, LS Houx, P Rasmussen, H Canet, J Rabbitt, P Jolles, J Larsen, K Hanning, CD Langeron, O Johnson, T Lauven, PM Kristensen, PA Biedler, A van Beem, H Fraidakis, O Silverstein, JH Beneken, JE Gravenstein, JS
Berridge MJ: Inositol trisphosphate and calcium signalling. Nature 1993; 361:315–25Berridge, MJ
Sedlak TW, Snyder SH: Messenger molecules and cell death: Therapeutic implications. JAMA 2006; 295:81–9Sedlak, TW Snyder, SH
Banerjee S, Hasan G: The InsP3 receptor: Its role in neuronal physiology and neurodegeneration. Bioessays 2005; 27:1035–47Banerjee, S Hasan, G
Lindholm D, Wootz H, Korhonen L: ER stress and neurodegenerative diseases. Cell Death Differ 2006; 13:385–92Lindholm, D Wootz, H Korhonen, L
Hanson CJ, Bootman MD, Roderick HL: Cell signalling: IP3 receptors channel calcium into cell death. Curr Biol 2004; 14:R933–5Hanson, CJ Bootman, MD Roderick, HL
Assefa Z, Bultynck G, Szlufcik K, Nadif KN, Vermassen E, Goris J, Missiaen L, Callewaert G, Parys JB, De Smedt H: Caspase-3-induced truncation of type 1 inositol trisphosphate receptor accelerates apoptotic cell death and induces inositol trisphosphate-independent calcium release during apoptosis. J Biol Chem 2004; 279:43227–36Assefa, Z Bultynck, G Szlufcik, K Nadif, KN Vermassen, E Goris, J Missiaen, L Callewaert, G Parys, JB De Smedt, H
Jayaraman T, Marks AR: T cells deficient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis. Mol Cell Biol 1997; 17:3005–12Jayaraman, T Marks, AR
Tang TS, Tu H, Chan EY, Maximov A, Wang Z, Wellington CL, Hayden MR, Bezprozvanny I: Huntingtin and Huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1. Neuron 2003; 39:227–39Tang, TS Tu, H Chan, EY Maximov, A Wang, Z Wellington, CL Hayden, MR Bezprozvanny, I
Paschen W, Mengesdorf T: Cellular abnormalities linked to endoplasmic reticulum dysfunction in cerebrovascular disease: Therapeutic potential. Pharmacol Ther 2005; 108:362–75Paschen, W Mengesdorf, T
Kindler CH, Eilers H, Donohoe P, Ozer S, Bickler PE: Volatile anesthetics increase intracellular calcium in cerebrocortical and hippocampal neurons. Anesthesiology 1999; 90:1137–45Kindler, CH Eilers, H Donohoe, P Ozer, S Bickler, PE
Madesh M, Hawkins BJ, Milovanova T, Bhanumathy CD, Joseph SK, Ramachandrarao SP, Sharma K, Kurosaki T, Fisher AB: Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis. J Cell Biol 2005; 170:1079–90Madesh, M Hawkins, BJ Milovanova, T Bhanumathy, CD Joseph, SK Ramachandrarao, SP Sharma, K Kurosaki, T Fisher, AB
Chan SL, Mayne M, Holden CP, Geiger JD, Mattson MP: Presenilin-1 mutations increase levels of ryanodine receptors and calcium release in PC12 cells and cortical neurons. J Biol Chem 2000; 275:18195–200Chan, SL Mayne, M Holden, CP Geiger, JD Mattson, MP
Guo Q, Sopher BL, Furukawa K, Pham DG, Robinson N, Martin GM, Mattson MP: Alzheimer’s presenilin mutation sensitizes neural cells to apoptosis induced by trophic factor withdrawal and amyloid beta-peptide: Involvement of calcium and oxyradicals. J Neurosci 1997; 17:4212–22Guo, Q Sopher, BL Furukawa, K Pham, DG Robinson, N Martin, GM Mattson, MP
Guo Q, Furukawa K, Sopher BL, Pham DG, Xie J, Robinson N, Martin GM, Mattson MP: Alzheimer’s PS-1 mutation perturbs calcium homeostasis and sensitizes PC12 cells to death induced by amyloid beta-peptide. Neuroreport 1996; 8:379–83Guo, Q Furukawa, K Sopher, BL Pham, DG Xie, J Robinson, N Martin, GM Mattson, MP
Trettel F, Rigamonti D, Hilditch-Maguire P, Wheeler VC, Sharp AH, Persichetti F, Cattaneo E, MacDonald ME: Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells. Hum Mol Genet 2000; 9:2799–809Trettel, F Rigamonti, D Hilditch-Maguire, P Wheeler, VC Sharp, AH Persichetti, F Cattaneo, E MacDonald, ME
Gines S, Ivanova E, Seong IS, Saura CA, MacDonald ME: Enhanced Akt signaling is an early pro-survival response that reflects N-methyl-D-aspartate receptor activation in Huntington’s disease knock-in striatal cells. J Biol Chem 2003; 278:50514–22Gines, S Ivanova, E Seong, IS Saura, CA MacDonald, ME
Franks NP, Lieb WR: Temperature dependence of the potency of volatile general anesthetics: Implications for in vitro  experiments. Anesthesiology 1996; 84:716–20Franks, NP Lieb, WR
Hiroi T, Wei H, Hough C, Leeds P, Chuang DM: Protracted lithium treatment protects against the ER stress elicited by thapsigargin in rat PC12 cells: Roles of intracellular calcium, GRP78 and Bcl-2. Pharmacogenomics J 2005; 5:102–11Hiroi, T Wei, H Hough, C Leeds, P Chuang, DM
Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260:3440–50Grynkiewicz, G Poenie, M Tsien, RY
Camello C, Lomax R, Petersen OH, Tepikin AV: Calcium leak from intracellular stores: The enigma of calcium signalling. Cell Calcium 2002; 32:355–61Camello, C Lomax, R Petersen, OH Tepikin, AV
Stutzmann GE: Calcium dysregulation, IP3 signaling, and Alzheimer’s disease. Neuroscientist 2005; 11:110–5Stutzmann, GE
Leissring MA, LaFerla FM, Callamaras N, Parker I: Subcellular mechanisms of presenilin-mediated enhancement of calcium signaling. Neurobiol Dis 2001; 8:469–78Leissring, MA LaFerla, FM Callamaras, N Parker, I
Gafni J, Munsch JA, Lam TH, Catlin MC, Costa LG, Molinski TF, Pessah IN: Xestospongins: Potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 1997; 19:723–33Gafni, J Munsch, JA Lam, TH Catlin, MC Costa, LG Molinski, TF Pessah, IN
Oka T, Sato K, Hori M, Ozaki H, Karaki H: Xestospongin C, a novel blocker of IP3 receptor, attenuates the increase in cytosolic calcium level and degranulation that is induced by antigen in RBL-2H3 mast cells. Br J Pharmacol 2002; 135:1959–66Oka, T Sato, K Hori, M Ozaki, H Karaki, H
De Smet P, Parys JB, Callewaert G, Weidema AF, Hill E, De Smedt H, Erneux C, Sorrentino V, Missiaen L: Xestospongin C is an equally potent inhibitor of the inositol 1,4,5-trisphosphate receptor and the endoplasmic-reticulum Ca2+ pumps. Cell Calcium 1999; 26:9–13De Smet, P Parys, JB Callewaert, G Weidema, AF Hill, E De Smedt, H Erneux, C Sorrentino, V Missiaen, L
Tang TS, Slow E, Lupu V, Stavrovskaya IG, Sugimori M, Llinas R, Kristal BS, Hayden MR, Bezprozvanny I: Disturbed Ca2+ signaling and apoptosis of medium spiny neurons in Huntington’s disease. Proc Natl Acad Sci U S A 2005; 102:2602–7Tang, TS Slow, E Lupu, V Stavrovskaya, IG Sugimori, M Llinas, R Kristal, BS Hayden, MR Bezprozvanny, I
Tang TS, Tu H, Orban PC, Chan EY, Hayden MR, Bezprozvanny I: HAP1 facilitates effects of mutant Huntingtin on inositol 1,4,5-trisphosphate-induced Ca release in primary culture of striatal medium spiny neurons. Eur J Neurosci 2004; 20:1779–87Tang, TS Tu, H Orban, PC Chan, EY Hayden, MR Bezprozvanny, I
Pan JZ, Xi J, Tobias JW, Eckenhoff MF, Eckenhoff RG: Halothane binding proteome in human brain cortex. J Proteome Res 2007; 6:582–92Pan, JZ Xi, J Tobias, JW Eckenhoff, MF Eckenhoff, RG
Matsuoka H, Kurosawa S, Horinouchi T, Kato M, Hashimoto Y: Inhalation anesthetics induce apoptosis in normal peripheral lymphocytes in vitro  . Anesthesiology 2001; 95:1467–72Matsuoka, H Kurosawa, S Horinouchi, T Kato, M Hashimoto, Y
Katayama T, Imaizumi K, Manabe T, Hitomi J, Kudo T, Tohyama M: Induction of neuronal death by ER stress in Alzheimer’s disease. J Chem Neuroanat 2004; 28:67–78Katayama, T Imaizumi, K Manabe, T Hitomi, J Kudo, T Tohyama, M
Xie Z, Moir RD, Romano DM, Tesco G, Kovacs DM, Tanzi RE: Hypocapnia induces caspase-3 activation and increases Aβ production. Neurodegener Dis 2004; 1:29–37Xie, Z Moir, RD Romano, DM Tesco, G Kovacs, DM Tanzi, RE
Xie ZC, Romano DM, Kovacs DM, Tanzi RE: Effects of RNA interference-mediated silencing of gamma-secretase complex components on cell sensitivity to caspase-3 activation. J Biol Chem 2004; 279:34130–7Xie, ZC Romano, DM Kovacs, DM Tanzi, RE
LaFerla FM: Calcium dyshomeostasis and intracellular signalling in Alzheimer’s disease. Nat Rev Neurosci 2002; 3:862–72LaFerla, FM
Planel E, Richter KE, Nolan CE, Finley JE, Liu L, Wen Y, Krishnamurthy P, Herman M, Wang L, Schachter JB, Nelson RB, Lau LF, Duff KE: Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia. J Neurosci 2007; 27:3090–7Planel, E Richter, KE Nolan, CE Finley, JE Liu, L Wen, Y Krishnamurthy, P Herman, M Wang, L Schachter, JB Nelson, RB Lau, LF Duff, KE
Kato R, Foex P: Myocardial protection by anesthetic agents against ischemia-reperfusion injury: An update for anesthesiologists. Can J Anaesth 2002; 49:777–91Kato, R Foex, P
Warner DS: Pharmacologic protection from ischemic neuronal injury. J Neurosurg Anesthesiol 2004; 16:95–7Warner, DS
Zuo Z, Wang Y, Huang Y: Isoflurane preconditioning protects human neuroblastoma SH-SY5Y cells against in vitro  simulated ischemia-reperfusion through the activation of extracellular signal-regulated kinases pathway. Eur J Pharmacol 2006; 542:84–91Zuo, Z Wang, Y Huang, Y
Bickler PE, Zhan X, Fahlman CS: Isoflurane preconditions hippocampal neurons against oxygen-glucose deprivation: Role of intracellular Ca2+ and mitogen-activated protein kinase signaling. Anesthesiology 2005; 103:532–9Bickler, PE Zhan, X Fahlman, CS
Bickler PE, Fahlman CS: The inhaled anesthetic, isoflurane, enhances Ca2+-dependent survival signaling in cortical neurons and modulates MAP kinases, apoptosis proteins and transcription factors during hypoxia. Anesth Analg 2006; 103:419–29Bickler, PE Fahlman, CS
Boyce M, Yuan J: Cellular response to endoplasmic reticulum stress: A matter of life or death. Cell Death Differ 2006; 13:363–73Boyce, M Yuan, J
Xu C, Bailly-Maitre B, Reed JC: Endoplasmic reticulum stress: Cell life and death decisions. J Clin Invest 2005; 115:2656–64Xu, C Bailly-Maitre, B Reed, JC
Wei H, Liang G, Yang H: Isoflurane preconditioning inhibited isoflurane-induced neurotoxicity. Neurosci Lett 2007; 425:59–62Wei, H Liang, G Yang, H
Matsumoto M, Nakagawa T, Inoue T, Nagata E, Tanaka K, Takano H, Minowa O, Kuno J, Sakakibara S, Yamada M, Yoneshima H, Miyawaki A, Fukuuchi Y, Furuichi T, Okano H, Mikoshiba K, Noda T: Ataxia and epileptic seizures in mice lacking type 1 inositol 1,4,5-trisphosphate receptor. Nature 1996; 379:168–71Matsumoto, M Nakagawa, T Inoue, T Nagata, E Tanaka, K Takano, H Minowa, O Kuno, J Sakakibara, S Yamada, M Yoneshima, H Miyawaki, A Fukuuchi, Y Furuichi, T Okano, H Mikoshiba, K Noda, T
Futatsugi A, Nakamura T, Yamada MK, Ebisui E, Nakamura K, Uchida K, Kitaguchi T, Takahashi-Iwanaga H, Noda T, Aruga J, Mikoshiba K: IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy metabolism. Science 2005; 309:2232–4Futatsugi, A Nakamura, T Yamada, MK Ebisui, E Nakamura, K Uchida, K Kitaguchi, T Takahashi-Iwanaga, H Noda, T Aruga, J Mikoshiba, K
Fig. 1. Inositol 1,4,5-trisphosphate (IP3) receptor total knock-out chicken lymphocytes display resistance to isoflurane-induced cell damage. The early cell damage was determined by the externalization of anionic phospholipid, phosphatidyl serine–annexin V binding (annexin V). The late cell damage was determined by propidium iodide (PI) staining, representing loss of plasma membrane integrity. Isoflurane induced cell damage dose-dependently (  A    C  ) and time-dependently (  D    F  ) in DT40 wild-type (WT) but not IP3receptor total knock-out cells. Representative imaging of annexin V–positive cells (  green  ) and PI-positive cells (  red  ) after treatment of isoflurane at various concentrations for 24 h (  A  ) or 2.4% isoflurane for various times (  D  ) in WT or IP3receptor total knock-out cells.  Scale bar  = 50 μm. Isoflurane for 24 h induced early (  B  ) and late (  C  ) cell damage dose-dependently in WT but not IP3receptor total knock-out cells. Isoflurane at 2.4% induced early (  E  ) and late (  F  ) cell damage time-dependently in WT but not IP3receptor total knock-out cells. Data represent mean ± SE from nine repeats of three separate experiments. ** or ***  P  < 0.01 or  P  < 0.001 compared with its corresponding control in WT cells. ###  P  < 0.001 compared with its own baseline before the isoflurane treatment. ISO = isoflurane; TKO = IP3receptor total knock-out. 
Image Not Available
Fig. 1. Inositol 1,4,5-trisphosphate (IP3) receptor total knock-out chicken lymphocytes display resistance to isoflurane-induced cell damage. The early cell damage was determined by the externalization of anionic phospholipid, phosphatidyl serine–annexin V binding (annexin V). The late cell damage was determined by propidium iodide (PI) staining, representing loss of plasma membrane integrity. Isoflurane induced cell damage dose-dependently (  A    C  ) and time-dependently (  D    F  ) in DT40 wild-type (WT) but not IP3receptor total knock-out cells. Representative imaging of annexin V–positive cells (  green  ) and PI-positive cells (  red  ) after treatment of isoflurane at various concentrations for 24 h (  A  ) or 2.4% isoflurane for various times (  D  ) in WT or IP3receptor total knock-out cells.  Scale bar  = 50 μm. Isoflurane for 24 h induced early (  B  ) and late (  C  ) cell damage dose-dependently in WT but not IP3receptor total knock-out cells. Isoflurane at 2.4% induced early (  E  ) and late (  F  ) cell damage time-dependently in WT but not IP3receptor total knock-out cells. Data represent mean ± SE from nine repeats of three separate experiments. ** or ***  P  < 0.01 or  P  < 0.001 compared with its corresponding control in WT cells. ###  P  < 0.001 compared with its own baseline before the isoflurane treatment. ISO = isoflurane; TKO = IP3receptor total knock-out. 
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Fig. 2. Inositol 1,4,5-trisphosphate (IP3) receptor total knock-out chicken lymphocytes were resistant to isoflurane-induced apoptosis and calcium elevations. (  A  ) Isoflurane at 2.4% for 24 h dramatically increased caspase-3 activity in wild-type (WT) but not IP3receptor total knock-out chicken lymphocytes. Data represent mean ± SE from three experiments. ***  P  < 0.001 compared with control. (  B  ) Isoflurane at 2.4% for 24 h significantly increased propidium iodide–positive cells (late cell damage) only in WT but not IP3receptor total knock-out cells, whereas thapsigargin (inhibitor of calcium adenosine triphosphatase on endoplasmic reticulum membrane as a positive control) still significantly increased propidium iodide–positive cells in both WT and IP3receptor total knock-out cells (n = 9; ***  P  < 0.001 compared with control). (  C  ) Representative tracing reveals rapid Ca2+mobilization followed by mitochondrial Ca2+uptake, as indicated by an increase in fluo-4 (  green  ) fluorescence and a subsequent increase in rhod-2 fluorescence in the mitochondria (  red  ) after exposure to 0.7 mm (2 minimum alveolar concentration) isoflurane or 1 μm thapsigargin. The same results were repeated in three separate experiments. Isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) precedes increase of [Ca2+]monly in WT (  C  ) but not in IP3receptor total knock-out cells (  D  ). Thapsigargin still significantly elevated both [Ca2+]cand mitochondria calcium concentration ([Ca2+]m) in IP3receptor total knock-out cells (  E  ). DT40 = chicken B lymphocytes; ISO = isoflurane; Tg = thapsigargin; TKO = IP3receptor total knock-out chicken B lymphocytes. 
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Fig. 2. Inositol 1,4,5-trisphosphate (IP3) receptor total knock-out chicken lymphocytes were resistant to isoflurane-induced apoptosis and calcium elevations. (  A  ) Isoflurane at 2.4% for 24 h dramatically increased caspase-3 activity in wild-type (WT) but not IP3receptor total knock-out chicken lymphocytes. Data represent mean ± SE from three experiments. ***  P  < 0.001 compared with control. (  B  ) Isoflurane at 2.4% for 24 h significantly increased propidium iodide–positive cells (late cell damage) only in WT but not IP3receptor total knock-out cells, whereas thapsigargin (inhibitor of calcium adenosine triphosphatase on endoplasmic reticulum membrane as a positive control) still significantly increased propidium iodide–positive cells in both WT and IP3receptor total knock-out cells (n = 9; ***  P  < 0.001 compared with control). (  C  ) Representative tracing reveals rapid Ca2+mobilization followed by mitochondrial Ca2+uptake, as indicated by an increase in fluo-4 (  green  ) fluorescence and a subsequent increase in rhod-2 fluorescence in the mitochondria (  red  ) after exposure to 0.7 mm (2 minimum alveolar concentration) isoflurane or 1 μm thapsigargin. The same results were repeated in three separate experiments. Isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) precedes increase of [Ca2+]monly in WT (  C  ) but not in IP3receptor total knock-out cells (  D  ). Thapsigargin still significantly elevated both [Ca2+]cand mitochondria calcium concentration ([Ca2+]m) in IP3receptor total knock-out cells (  E  ). DT40 = chicken B lymphocytes; ISO = isoflurane; Tg = thapsigargin; TKO = IP3receptor total knock-out chicken B lymphocytes. 
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Fig. 3. The inositol 1,4,5-trisphosphate (IP3) receptor antagonist xestospongin C (Xc) inhibited isoflurane-induced cytotoxicity and calcium elevations in PC12 cells transfected with L286V presenilin 1. (  A  ) Representative images of phase contrast (PC) or propidium iodide (PI) staining of L286V or wild-type (WT) PC12 cells immediately after treatment of 2.4% isoflurane for 24 h.  Scale bar  = 100 μm. (  B  ) Quantification of percentage of PI-positive cells (late cell damage) after treating L286V or WT PC12 cells with 2.4% isoflurane for 24 h. L286V PC12 cells were more vulnerable than WT PC12 cells to isoflurane-induced cytotoxicity. Data represent mean ± SE from 25 repeats of three separate experiments. *** or ###  P  < 0.001 compared with control in L286V or isoflurane treatment in WT, respectively. (  C  ) Pretreatment of 100 nm Xc for 30 min abolished the lactate dehydrogenase (LDH) release (late cell damage) induced by 2.4% isoflurane for 24 h in L286V cells. Data represent mean ± SE from 12 repeats of three separate experiments. **  P  < 0.01 compared with control. ##  P  < 0.01 compared with isoflurane treatment alone. (  D  ) Representative tracing reveals averaged isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) with or without pretreatment of Xc in the absence of extracellular calcium in both WT and L286V PC12 cells. (  E  ) Isoflurane at 2.4% induced significantly higher peak elevation of [Ca2+]cin the absence of extracellular calcium in L286V than in WT PC12 cells. Xc (1 μm) significantly inhibited isoflurane-induced peak elevation of [Ca2+]cin the absence of extracellular calcium in both WT and L286V cells. Data represent mean ± SE from three separate experiments. * or **  P  < 0.05 or  P  < 0.01 compared with isoflurane treatment alone without Xc pretreatment. ##  P  < 0.01 compared with WT PC12 cells treated with isoflurane alone. ISO = isoflurane; L286V = PC12 cells with presenilin-1 mutation. 
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Fig. 3. The inositol 1,4,5-trisphosphate (IP3) receptor antagonist xestospongin C (Xc) inhibited isoflurane-induced cytotoxicity and calcium elevations in PC12 cells transfected with L286V presenilin 1. (  A  ) Representative images of phase contrast (PC) or propidium iodide (PI) staining of L286V or wild-type (WT) PC12 cells immediately after treatment of 2.4% isoflurane for 24 h.  Scale bar  = 100 μm. (  B  ) Quantification of percentage of PI-positive cells (late cell damage) after treating L286V or WT PC12 cells with 2.4% isoflurane for 24 h. L286V PC12 cells were more vulnerable than WT PC12 cells to isoflurane-induced cytotoxicity. Data represent mean ± SE from 25 repeats of three separate experiments. *** or ###  P  < 0.001 compared with control in L286V or isoflurane treatment in WT, respectively. (  C  ) Pretreatment of 100 nm Xc for 30 min abolished the lactate dehydrogenase (LDH) release (late cell damage) induced by 2.4% isoflurane for 24 h in L286V cells. Data represent mean ± SE from 12 repeats of three separate experiments. **  P  < 0.01 compared with control. ##  P  < 0.01 compared with isoflurane treatment alone. (  D  ) Representative tracing reveals averaged isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) with or without pretreatment of Xc in the absence of extracellular calcium in both WT and L286V PC12 cells. (  E  ) Isoflurane at 2.4% induced significantly higher peak elevation of [Ca2+]cin the absence of extracellular calcium in L286V than in WT PC12 cells. Xc (1 μm) significantly inhibited isoflurane-induced peak elevation of [Ca2+]cin the absence of extracellular calcium in both WT and L286V cells. Data represent mean ± SE from three separate experiments. * or **  P  < 0.05 or  P  < 0.01 compared with isoflurane treatment alone without Xc pretreatment. ##  P  < 0.01 compared with WT PC12 cells treated with isoflurane alone. ISO = isoflurane; L286V = PC12 cells with presenilin-1 mutation. 
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Fig. 4. Xestospongin C (Xc) inhibited isoflurane-induced cytotoxicity and calcium elevations in mutated Huntington disease (HD) knocked-in striatal cells. (  A  ) Representative images of phase contrast (PC) or propidium iodide (PI) staining of HD or wild-type (WT) striatal cells at 24 h after completion of treating cells with 2.4% isoflurane for 24 h.  Scale bar  = 100 μm. (  B  ) Isoflurane at 2.4% for 24 h significantly increased percentage of cell damage determined by PI staining in HD knocked-in but not in WT striatal cells. Data represent mean ± SE of 18 repeats from three separate experiments. *  P  < 0.05 compared with control in HD striatal cells. (  C  ) Pretreatment of 100 nm Xc for 30 min abolished the MTS reduction induced by 2.4% isoflurane for 24 h in HD striatal cells. Data represent mean ± SE from 12 repeats of three separate experiments. *** or ###  P  < 0.001 compared with control or isoflurane treatment alone. (  D  ) Representative tracing reveals averaged isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) with or without pretreatment of Xc in the absence of extracellular calcium in both WT and HD cells. (  E  ) Isoflurane at 2.4% induced significantly higher peak elevation of [Ca2+]cin the absence of extracellular calcium in HD knocked-in than in WT striatal cells. Xc (1 μm) nearly abolished isoflurane-induced peak elevation of [Ca2+]cin the absence of extracellular calcium in both WT and HD striatal cells. Data represent mean ± SE from three separate experiments. **  P  < 0.01 compared with isoflurane treatment alone without Xc pretreatment. ##  P  < 0.01 compared with WT striatal cells treated with isoflurane alone. ISO = isoflurane; MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. 
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Fig. 4. Xestospongin C (Xc) inhibited isoflurane-induced cytotoxicity and calcium elevations in mutated Huntington disease (HD) knocked-in striatal cells. (  A  ) Representative images of phase contrast (PC) or propidium iodide (PI) staining of HD or wild-type (WT) striatal cells at 24 h after completion of treating cells with 2.4% isoflurane for 24 h.  Scale bar  = 100 μm. (  B  ) Isoflurane at 2.4% for 24 h significantly increased percentage of cell damage determined by PI staining in HD knocked-in but not in WT striatal cells. Data represent mean ± SE of 18 repeats from three separate experiments. *  P  < 0.05 compared with control in HD striatal cells. (  C  ) Pretreatment of 100 nm Xc for 30 min abolished the MTS reduction induced by 2.4% isoflurane for 24 h in HD striatal cells. Data represent mean ± SE from 12 repeats of three separate experiments. *** or ###  P  < 0.001 compared with control or isoflurane treatment alone. (  D  ) Representative tracing reveals averaged isoflurane-evoked elevation of cytosolic calcium concentration ([Ca2+]c) with or without pretreatment of Xc in the absence of extracellular calcium in both WT and HD cells. (  E  ) Isoflurane at 2.4% induced significantly higher peak elevation of [Ca2+]cin the absence of extracellular calcium in HD knocked-in than in WT striatal cells. Xc (1 μm) nearly abolished isoflurane-induced peak elevation of [Ca2+]cin the absence of extracellular calcium in both WT and HD striatal cells. Data represent mean ± SE from three separate experiments. **  P  < 0.01 compared with isoflurane treatment alone without Xc pretreatment. ##  P  < 0.01 compared with WT striatal cells treated with isoflurane alone. ISO = isoflurane; MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. 
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