A large body of recent work indicates that volatile anesthetics, particularly sevoflurane, protect the brain against ischemic injury in various experimental models.1–4 It was also recently shown that sevoflurane preconditions the rat hippocampus against ischemic damage.5,6 However, the relevance of these findings to the perioperative context remains unproven. In a model of focal cerebral ischemia in vivo, isoflurane has been shown to protect the brain against both cell death and infarction, but this protecting effect was only transient.7 In this study, the decay in isoflurane neuroprotection could be attributed to the inability of this drug to inhibit long-term apoptosis. Also, the duration of ischemia (50 and 80 min of mean cerebral artery occlusion) was a critical factor for the neuroprotective properties of isoflurane. In a model of incomplete hemispheric ischemia (focal ischemia with hemorrhagic hypotension), it was shown that sevolurane exerted a sustained neuroprotective effect up to 28 days postinjury.8 Interestingly, both necrotic and apoptotic cell death were attenuated by sevoflurane in these experiments. Although experimental conditions were different, the duration of ischemia might have also contributed to the explanation, in part, of the differences observed between the neuroprotective actions of isoflurane and sevoflurane in these studies.
We have previously shown that short exposure to volatile anesthetics and dexmedetomidine increases phosphorylation of the nonreceptor tyrosine kinase focal adhesion kinase (FAK, a key enzyme of cell signaling involved in some long lasting changes in the central nervous system).9,10 Therefore, in the present study, we used the rat acute oxygen-glucose deprived (OGD) in vitro hippocampal slice to investigate: 1) whether the protective effect of a preconditioning concentration of sevoflurane depends on the duration of the ischemic insult; 2) and whether FAK downstream signal is involved in this effect.
Experiments were performed on 1-mo-old male Sprague-Dawley rats (Iffa-Credo, France) weighing 250 g and housed on a 12:12 light/dark cycle with food and water ad libitum. Approval was obtained from the Institutional Care and Use Committee at Paris 7 University (Paris, France). This study, including care of animals involved, was conducted according to the official edict presented by the French Ministry of Agriculture (Paris, France). Therefore, these experiments were conducted in an authorized laboratory and supervised by an authorized researcher (Pierre Gressens, MD, PhD, INSERM U 676, Paris France).
Experimental Protocol and Measurement of Phosphorylated FAK Expression
Preparation of hippocampal slices has been reported in detail elsewhere.9,10 Briefly, hippocampal slices (300 μm thickness) were incubated 60 min with 1 mL Ca2+ -free artificial cerebrospinal fluid (aCSF, 60 min, 37°C) containing 126.5 mM NaCl, 27.5 mM NaHCO3, 2.4 mM KCl, 0.5 mM KH2PO4, 1.93 mM MgCl2, 0.5 mM Na2SO4, 4 mM glucose and 11 mM HEPES adjusted to pH 7.4 with 95%/5% [vol/vol] oxygen/carbon dioxide (CO2) mixture. Tetrodotoxin (TTX, 1 μM) was added at this time point to avoid indirect effects due to neuronal firing on FAK phosphorylation. Slices were then transferred to air-tight chambers (1 cm3 volume, 10 slices per chamber) and superfusion at 10 mL/min during 3 h in an oxygenated CSF containing: 126.5 mM NaCl, 27.5 mM NaHCO3, 2.4 mM KCl, 0.5 mM KH2PO4, 1.93 mM MgCl2, 0.5 mM Na2SO4, 4 mM glucose, CaCl2 0.5 mM, and 11 mM HEPES adjusted to pH 7.4. The slices were then superfused during 10, 20, 30, 45, 55, and 60 min with either oxygenated CSF (control) or a glucose-free CSF bubbled with 95%N2–5%CO2 containing dithionite (1 mM, Sigma), an O2 absorbent (glucose oxygen deprivation). Temperature, pH, partial pressure of O2, and CO2 were closely monitored. Temperature in the chambers was servocontrolled to 37°. In both control and hypoxic aglycemic conditions, sevoflurane dissolved in dimethyl sulfoxide (DMSO, Sigma) at 1:100 dilution was administered as a preconditioning challenge for 1 h. Attention was paid to minimize sevoflurane evaporation. Concentration of sevoflurane in the medium was checked after 30 min of incubation using gas phase chromatography according to a slightly modified version of the method of Brachet-Liermain et al., as previously reported and routinely used in our laboratory.9 The Scr inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2, 10−5 M) was added 60 min before that of sevoflurane and maintained throughout incubation with sevoflurane. Slices were then transferred in oxygenated CSF for 3 h until the various periods of OGD (10, 20, 30, 45, 55, and 60 min) were initiated. At the end of the OGD period, CSF was analyzed for pH, Po2, and Pco2 (Radiometer Copenhagen, ABL700, DK-2700 Brønshøj, Denmark). Slices were recovered in oxygenated buffered CSF containing 4 mM glucose for 1 h, since this delay provided appropriate conditions for expression of cleaved caspase 3 in the preparation.10,11 At the end of reperfusion, slices designated to Western blot analysis were frozen in liquid nitrogen and homogenized by sonication in 200 μL of a solution of 1% (wt/vol) sodium dodecyl sulfate, 1 mM sodium orthovanadate and antiproteases (50 μg/mL leupeptin, 10 μg/mL aprotinin and 5 μg/mL pepstatin, Sigma) in water at 100°C. Homogenates were stored at −80°C until processing. Protein concentration in the homogenates was determined with a bicinchoninic acid-base method, using bovine serum albumin as the standard. Equal amounts of protein (30 μg) were subjected to 13% polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and transferred electrophoretically to nitrocellulose. Primary antibodies were labeled with peroxidase-coupled antibodies against rabbit Immunoglobulin G, which were detected by exposure of MP autoradiographic films in the presence of a chemiluminescent reagent (ECL, Amersham, Little Chalfont, UK). The specificity of the immunoreactivity for FAK was assessed by its competition in the presence of 50 μM-O-phosphotyrosine. Identification of phosphorylated FAK was performed using a rabbit anti-Y397 FAK phosphospecific antibody (Biosource International diluted 1:1000). Identification of total FAK was made with an anti-FAK antibody directed against the nonphosphorylated residues of the protein (Biosource International, Camarillo, CA: diluted 1:1000). The same methodology was used to determine β-actin protein expression (quantified by using the specific monoclonal antiactin A5316 antibody, Sigma). Immunoreactive bands were quantified using a computer-assisted densitometer and normalized to total FAK expression (Cohu High Performance CCD camera, Gel Analyst 3.01 pci, Paris, France). For each band, blank values were subtracted before calculating the ratios.
Quantification of Cell Death and Sevoflurane Neuroprotection
Quantification of cell death was performed using two methods: detection of active cleaved caspase 3 by Western blot analysis and propidium iodide (PI) fluorescence. For detection of active caspase 3, immunoblot analysis was performed with the rabbit polyclonal Immunoglobulin G anti-caspase 3 specific antibodies detecting both the 32 KDa entire protein and the 17 KDa fragment produced by cleavage of caspase 3 when activated, as previously used in our laboratory (Upstate biotechnology diluted 1:2000, Euromedex Souffelweyersheim, France). The 17 Kda band immunoreactivity was considered cleaved caspase 3 and considered in the statistical analysis. Immunoreactive bands normalized to β-actin band were quantified using specific monoclonal antiactin A5316 antibody (Sigma) using a computer-assisted densitometer and expressed as a percentage of increase from control. For each band, blank values were subtracted before calculating the ratios.
Quantification of cell death and sevoflurane neuroprotection were also detected using fluorescent PI in the CA1 subfield area of hippocampus (Invitrogen, Cergy Pontoise, France, P-3566.10,12 PI binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per four to five pairs of DNA bases. Once the dye is bound to nucleic acids, its fluorescence is enhanced 20- to 30-fold, the maximal absorption wave for PI being 535 nm and the maximal fluorescence emission wave being 617 nm. PI was added in the CSF (3 μM) from the beginning of OGD to the end of the experiment in 3 independent experiments. In each of them, 5 slices were randomly selected and processed for serial 100-μm coronal sections cut along the entire hippocampus. Stained cells were examined using a fluorescence microscope equipped with an appropriate filter (UV-2A; Zeiss, Oberkochen, Germany; excitation, 530 nm; emission, >600 nm) and images were digitalized. All the slices were analyzed at the same time by an observer unaware of the treatment assignment. For each slice, PI fluorescence of 10 areas from the CA1 subfield were analyzed using Image J 1.31 v software.
For Western blot analysis, data were collected from 10 independent experiments run in triplicate. Each independent experiment was performed with 1 animal (n = 30). For PI fluorescence, 3 independent experiments were used for each condition. In each experiment, fluorescence from 10 random areas were analyzed in 5 slices (n = 150 for each condition). After the normality of data was accessed, statistical analysis was performed by analysis of variance followed by post hoc analysis using Student's t-test with Bonferroni correction. Results are expressed as mean ± sd. P < 0.05 was considered the threshold for significance.
Po2, Pco2, and pH in the hynoxic aglycemic versus control chambers were 3 ± 2 vs 440 ± 18 mm Hg (P < 0.05), 41 ± 3 vs 41 ± 2 mm Hg (NS) and 7.4 ± 0.01 vs 7.4 ± 0.02 (NS). PI fluorescence increased between 20 and 30 min of OGD. This effect reached statistical significance at 20 and 30 min, but not at 10 min, and was still observed for 45, 50, and 60 min of OGD (Fig. 1). Protein expression of active caspase 3 significantly increased from 10 to 45 min in comparison with control conditions, remained stable at 45 and 50 min and decreased at 60 min of OGD (Fig. 2).
In control conditions, sevoflurane 10−4 M alone did not alter active caspase 3 expression and PI fluorescence (Figs. 1 and 2). Sevoflurane (10−4 M) increased the protein expression of phosphorylated FAK normalized to total FAK by 249 ± 63.5 (P < 0.0001, Fig. 3), whereas β-Actin immunoreactivity was unchanged under OGD conditions. A preconditioning application of sevoflurane (10−4 M, 1 h, 3 h before onset of OGD) significantly decreased caspase 3 expression for OGD durations of 10, 20, and 30 min, as well as PI fluorescence at 20 and 30 min. In contrast, sevoflurane failed to decrease caspase 3 expression and PI fluorescence for ischemia durations of 45, 50, and 60 min (Figs. 1 and 2). In control conditions, DMSO (1:100) alone did not affect caspase 3 expression (103% ± 22%, nonsignificant versus control) nor PI fluorescence (108 ± 11, nonsignificant versus control). DMSO also had no effect on both PI fluorescence (102 ± 33, 4032 ± 840, 6034 ± 1933, 6300 ± 1143, 5844 ± 1397, 6020 ± 1068, at 10, 20, 30, 45, 50, and 60 min of OGD, respectively, nonsignificant versus OGD alone) or active caspase 3 expression (106 ± 22, 242 ± 57, 403 ± 124, 497 ± 158, 412 ± 73, 278 ± 79 at 10, 20, 30, 45, 50, and 60 min of OGD respectively, nonsignificant versus OGD alone).
Preincubation of slices with PP2 60 min before and throughout sevoflurane incubation and OGD (30 min) significantly decreased the neuroprotective effect of sevoflurane on cell death (caspase 3 activation [Figs. 1 and 2]). PP2 per se had no effect on caspase 3 activation and PI fluorescence in both control and OGD (Figs. 4 and 5). The preconditioned effects of sevoflurane on both caspase 3 expression and cell death labeled with PI were nonsignificantly different in the presence or absence of TTX (1 μM, Figs. 4 and 5).
The main original findings of the present study can be summarized as follows: a clinical sevoflurane concentration exerts a preconditioning effect against ischemic brain injury in the OGD hippocampal slice. This effect consists of significantly attenuating the increase in active caspase 3 (a key enzyme of the apoptotic cascade) expression for ischemia durations equal to or shorter than 30 min. This effect is very likely to involve phosphorylation of the nonreceptor tyrosine kinase FAK.
We observed a nonlinear increase in PI fluorescence with the duration of ischemia. Fluorescence was not significantly different from control conditions for a 10 min ischemic period. A statistically significant difference was achieved at 20 and 30 min, followed by a ceiling effect until 60 min of ischemia. Significant cell death has been reported after 10 min of ischemia in the CA1 area using a similar approach.13 This difference might be explained either by the high superfusion rate used in our study (10 mL/min) or the shorter duration of the reperfusion period (1 vs 6 h) that likely contributed to a decrease in the amount of excitotoxic glutamate present in the slice and delayed the occurrence of cell death. Active caspase 3 protein expression was found significantly increased at both times of OGD, with an increase from 10 to 45 min followed by a decrease from 50 to 60 min of OGD. The time courses of PI fluorescence and active caspase 3 expression are consistent (with, but do not prove, the development of apoptotic cell death for moderate ischemic stimuli. We did not find any toxic effect of sevoflurane in normoxic normoglycemic conditions neither by analyzing PI fluorescence in CA1 nor by studying caspase 3. This finding is different from that reported by Zhan et al. 14 in the same model of the acute rat hippocampal slice in which a neurotoxic effect of isoflurane (1%, 20 min) per se was reported. Cell death was observed in all hippocampus subfields in older rats (24 mo), but not in younger ones (5-days-old). Several hypotheses may account for these apparent discrepancies: first, toxicity of volatile anesthetics on neuronal cells reported in 24-mo-aged animals may not be present in rats aged 1 mo, as we used in our study. Second, slices were continuously superfused at a 10 mL/min rate in our experiments. This probably contributed to reducing the amount of excitotoxicity, thereby reducing the potential for excitotoxic cell injury. Third, sevoflurane and isoflurane may behave differently and differentially affect cell survival in the hippocampus. Finally, the organotypic slice culture would probably be more appropriate for studying anesthetic-induced neurotoxicity, since a 6 h exposure to isoflurane has been recently reported to induce apoptosis in neuroglioma cells.15 In our study, sevoflurane preconditioning significantly attenuated both expression of active caspase 3 and PI fluorescence. Interestingly, sevoflurane failed to affect both active caspase 3 expression and PI fluorescence when duration of OGD was more than 30 min. This suggests that sevoflurane's neuroprotective effect depends on the duration of ischemia applied to the OGD hippocampal slice. More generally, further increasing the severity of ischemia via an increase in the duration of ischemia may surpass the ability of anesthetics to decrease cell death and apoptosis.
The major mechanisms generally considered to be involved in volatile anesthetic-neuroprotection are the decrease in glutamate excitotoxicity, reduction in ATP depletion, γ-aminobutyric acid-A receptor potentiation16 or reduction in catecholamines and excitotoxic transmitters (such as glutamate) release.3,4 The implication of these neuroprotective mechanisms in the preconditioning effect of sevoflurane observed in the current work remains to be investigated. Volatile anesthetics have been shown to produce both neuroprotection and preconditioning through cellular pathways involving protein kinase C (PKC), nitric oxide, mitogen-activated kinases, and PI3K-Akt pathways.17,18 Our findings confirm and extend our previous results showing that sevoflurane stimulates the phosphorylation of FAK, a key enzyme in cellular signaling, which plays a role in plasticity and survival.19 This effect depends, in hippocampal slices, on the activation of PKC.9 These findings are consistent with previous results showing that sevoflurane immediate preconditioning is sensitive to chelerythrin, a PKC-protein kinase M inhibitor.6 Alternatively, sevoflurane-induced activation of reactive oxygen species (ROS) has been shown in cardiac tissue.20 Whether these findings may be extrapolated to the central nervous system remain to be determined. However, sevoflurane has been shown to activate FAK phosphorylation through a pathway depending on phospholipase and PKC activation, whereas ROS has been shown to activate FAK directly or via modulation of phosphatase activities.21–23 Therefore, it cannot be excluded that ROS production might directly or indirectly play a role in the preconditioning effects of sevoflurane reported here. Increasing FAK phosphorylation correlates with attenuation of cell death induced by a preconditioning application of dexmedetomidine in the hippocampal slice.10 Here, PP2 significantly inhibited both PI and caspase 3 decrease induced by sevoflurane preconditioning. PP2 is a highly specific inhibitor of Src kinases involved in the signaling cascades initiated by FAK phosphorylation in various tissues, including the brain.19 Altogether, these results support that activation of FAK is very likely to be involved in the preconditioning effect of sevoflurane reported for durations of ischemia <30 min.
Our study also indicates that sevoflurane may activate antiapoptotic factors together with increasing FAK phosphorylation. Indeed, a sustained reduction in neuronal damage has been observed after incomplete cerebral ischemia with reperfusion in rats anesthetized with sevoflurane.8 In this study, although it focused on per-insult neuroprotection and not preconditioning, a marked decrease in active caspase 3 expression was reported.8 This is likely explained by the increase by sevoflurane of the concentration of the anti-apoptotic protein Bcl-2. FAK and Src kinases activation has been shown to modulate the apoptotic cascade by inhibiting BAD proaoptotic protein through Akt stimulation.19 Bickler et al. have shown isoflurane preconditioning to activate this protein in a model of organotypic slice culture.24 This mechanism may be transposed to our findings as well and might explain the reduction of active caspase 3 expression by a preconditioning concentration of sevoflurane.
Our study certainly has limitations. The OGD hippocampal slice represents a robust, reliable in vitro model to examine the role of pharmacologic interventions modulating ischemic injury of brain tissue.10,13 Energy deprivation in hippocampal slices results in a cascade of events triggered by excitotoxic glutamate and free radicals leading to neuronal necrosis and apoptotic death.13,25,26 Unlike the organotypic slice culture or other in vivo models such as incomplete hemispheric ischemia, the OGD hippocampal slice allows examination of only early events in this cascade, which is a limitation of this model (27–29). Control of Po2, Pco2, and pH in the superfusion chambers was satisfactory, since Po2 of <5 mm Hg was obtained in the anoxic compartment. It could be argued that the use of TTX, a drug exhibiting neuroprotective neuroproperties in some models, limits the interpretation of the data obtained with sevoflurane.18 The use of TTX was necessary to avoid neuronal firing when measuring FAK phosphorylation. However, sevoflurane's effects on caspase 3 expression were similar whether TTX was present. The sevoflurane concentration used here (10−4 M) was close to an equivalent 2% inhaled sevoflurane concentration (1 minimum alveolar anesthetic concentration-equivalent concentration), which is clearly within the clinical range. In a previous study, we have shown this concentration to be the EC50 value for sevoflurane-induced FAK phosphorylation.9 We measured cell death by PI fluorescence and active caspase 3 protein expression. PI fluorescence is a valid morphologic indicator of cell death.12,28 We focused on the CA1 hippocampus subfield area because of its particular sensitivity to ischemia in comparison to other areas such as dentate gyrus.24 We do not exclude, however, that the effects of sevoflurane reported in the CA1 area may be not identical to those in the DG or CA3 areas. Although we did not perform extensive measurements of apoptotic cell death, the increase in the expression of the 17 kda fragment of caspase 3, a key enzyme in the production of apoptotic death, suggests that caspase 3 was cleaved and activated by OGD. Brain caspase 3 expression has been found to be increased in neuronal cells when ischemia or hypoperfusion was present.8 Therefore, apoptotic cell death was likely to be present in the slices subjected to ischemia and reperfusion. Dithionite was used in our experiments to decrease O2 tension during the OGD periods. This compound has been shown to produce superoxyde radicals and affect neurons independently of O2 deprivation.30 Therefore, we cannot exclude that dithionite per se may have affected cell survival under OGD conditions.
In conclusion, we have shown that sevoflurane exerts a preconditioning effect against ischemic cell death in the rat acute hippocampal slice, depending on both tyrosine kinases and duration of ischemia. Therefore, the duration of ischemia may represent a critical factor to account for the variability in the neuroprotective efficacy of anesthetics in experimental models.
1. Warner DS, McFarlane C, Todd MM, Ludwig P, McAllister AM. Sevoflurane and halothane reduce focal ischemic brain damage in the rat. Possible influence on thermoregulation. Anesthesiology 1993;79:985–92
2. Werner C, Mollenberg O, Kochs E, Schulte J am Esch. Sevoflurane improves neurological outcome after incomplete cerebral ischaemia in rats. Br J Anaesth 1995;75:756–60
3. Toner CC, Connelly K, Whelpton R, Bains S, Michael-Titus AT, McLaughlin DP, Stamford JA. Effects of sevoflurane on dopamine, glutamate and aspartate release in an in vitro model of cerebral ischaemia. Br J Anaesth 2001;86:550–4
4. Englelhard K, Werner C, Hoffman WE, Matthes B, Blobner M, Kochs E. The effect of sevoflurane and propofol on cerebral neurotransmitter concentrations during cerebral ischemia in rats. Anesth Analg 2003;97:1155–61
5. Lei B, Popp S, Capuano-Waters C, Cottrell JE, Kass IS. Sevoflurane preconditioning protects rat hippocampus against ischemic neuronal damage. J Neurosurg Anesthesiol 2006;18:286–7
6. Wang J, Lei B, Popp S, Meng F, Cottrell JE, Kass IS. Sevoflurane immediate preconditioning alters hypoxic membrane potential changes in rat hippocampal slices and improves recovery of CA1 pyramidal cells after hypoxia and global cerebral ischemia. Neuroscience 2007;145:1097–107
7. Kawaguchi M, Kimbro JR, Drummond JC, Cole DJ, Kelly PJ, Patel PM. Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia. Anesthesiology 2000;92:1335–42
8. Pape M, Englelhard K, Eberspacher E, Hollweck R, Kellermann K, Zintner S, Hutzler P, Werner C. The long-term effect of sevoflurane on neuronal cell damage and expression of apoptotic factors after cerebral ischemia and reperfusion in rats. Anesth Analg 2006;103:173–9
9. Dahmani S, Tesniere A, Rouelle D, Toutant M, Desmonts JM, Mantz J. Effects of anesthetic agents on focal adhesion kinase (pp125FAK) tyrosine phosphorylation in rat hippocampal slices. Anesthesiology 2004;101:344–53
10. Dahmani S, Rouelle D, Gressens P, Mantz J. Effects of dexmedetomidine on hippocampal focal adhesion kinase tyrosine phosphorylation in physiologic and ischemic conditions. Anesthesiology 2005;103:969–77
11. Dahmani S, Rouelle D, Gressens P, Mantz J. The effects of lidocaine and bupivacaine on protein expression of cleaved caspase 3 and tyrosine phosphorylation in the rat hippocampal slice. Anesth Analg 2007;104:119–23
12. Laake JH, Haug FM, Wieloch T, Ottersen OP. A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence. Brain Res Brain Res Protoc 1999;4:173–84
13. Popovic R, Liniger R, Bickler PE. Anesthetics and mild hypothermia similarly prevent hippocampal neuron death in an in vitro model of cerebral ischemia. Anesthesiology 2000;92:1343–9
14. Zhan X, Fahlman CS, Bickler PE. Isoflurane neuroprotection in rat hippocampal slices decreases with aging: changes in intracellular Ca2+ regulation and N
-methyl-d-aspartate receptor-mediated Ca2+ influx. Anesthesiology 2006;104:995–1003
15. 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;104:988–94
16. Elsersy H, Mixco J, Sheng H, Pearlstein RD, Warner DS. Selective gamma-aminobutyric acid type A receptor antagonism reverses isoflurane ischemic neuroprotection. Anesthesiology 2006;105:81–90
17. Wang L, Traystman RJ, Murphy SJ. Inhalational anesthetics as preconditioning agents in ischemic brain. Curr Opin Pharmacol 2008;8:104–10
18. Kitano H, Kirsch JR, Hurn PD, Murphy SJ. Inhalational anesthetics as neuroprotectants or chemical preconditioning agents in ischemic brain. J Cereb Blood Flow Metab 2007;27:1108–28
19. Girault JA, Costa A, Derkinderen P, Studler JM, Toutant M. FAK and PYK2/CAKbeta in the nervous system: a link between neuronal activity, plasticity and survival? Trends Neurosci 1999;22:257–63
20. Kevin LG, Novalija E, Riess ML, Camara AK, Rhodes SS, Stowe DF. Sevoflurane exposure generates superoxide but leads to decreased superoxide during ischemia and reperfusion in isolated hearts. Anesth Analg 2003;96:949–55, table of contents
21. Vepa S, Scribner WM, Parinandi NL, English D, Garcia JG, Natarajan V. Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells. Am J Physiol 1999;277:L150–L158
22. Hao Q, Rutherford SA, Low B, Tang H. Suppression of the phosphorylation of receptor tyrosine phosphatase-alpha on the Src-independent site tyrosine 789 by reactive oxygen species. Mol Pharmacol 2006;69:1938–44
23. Seo JH, Ahn Y, Lee SR, Yeol Yeo C, Chung Hur K. The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway. Mol Biol Cell 2005;16:348–57
24. 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;103:532–9
25. Larsen M, Haugstad TS, Berg-Johnsen J, Langmoen IA. The effect of isoflurane on brain amino acid release and tissue content induced by energy deprivation. J Neurosurg Anesthesiol 1998;10:166–70
26. Kass IS, Amorim P, Chambers G, Austin D, Cottrell JE. The effect of isoflurane on biochemical changes during and electrophysiological recovery after anoxia in rat hippocampal slices. J Neurosurg Anesthesiol 1997;9:280–6
27. Patel P. No magic bullets: the ephemeral nature of anesthetic-mediated neuroprotection. Anesthesiology 2004;100:1049–51
28. Sullivan BL, Leu D, Taylor DM, Fahlman CS, Bickler PE. Isoflurane prevents delayed cell death in an organotypic slice culture model of cerebral ischemia. Anesthesiology 2002; 96:189–95
29. Feiner JR, Bickler PE, Estrada S, Donohoe PH, Fahlman CS, Schuyler JA. Mild hypothermia, but not propofol, is neuroprotective in organotypic hippocampal cultures. Anesth Analg 2005;100:215–25
© 2009 International Anesthesia Research Society
30. Gebhardt C, Heinemann U. Anoxic decrease in potassium outward currents of hippocampal cultured neurons in absence and presence of dithionite. Brain Res 1999;837:270–6