1 Department of Anaesthesia, Bichat University Hospital, 46 rue Henri Huchard, F-75018 Paris, France. 2 Institut National de la Santé et de la Recherche Médicale (INSERM) E9935, Robert Debré University Hospital, 40 Bd Sérurier, F-75019 Paris, France
* Corresponding author. E-mail: jean.mantz{at}bch.ap-hop-paris.fr
Accepted for publication March 12, 2004.
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Abstract |
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Methods. Rathippocampal slices were subjected to an anoxic-aglycaemic (or physiologic, control) challenge followed by 3-h reperfusion, and treated with various concentrations of thiopental and isoflurane. PP125FAK phosphorylation was measured by immunoblotting. Neuronal death was assessed by immunostaining with bis-benzimide.
Results. Significant neuronal death was detected after 30 min (but not 10) of anoxia-aglycaemia (40 (4) vs 14 (5)% of control, P<0.05). At 30 min, phosphorylated pp125FAK content was significantly decreased by anoxic glucose-free conditions (55 (27)% of control, P<0.05). This effect was markedly attenuated by thiopental (10 and 100 µM) and isoflurane (1 and 2%). Under control conditions, thiopental (1, 10, and 100 µM) and isoflurane (0.5, 1, and 2%) increased pp125FAK phosphorylation in a concentration-related fashion. This effect was blocked by chelerythrin and bisindolylmaleimide I and IX (10 µM, three structurally distinct inhibitors of protein kinase C, PKC) but not the N-methyl-D-aspartate (NMDA) receptor antagonist MK801 (10 µM).
Conclusion. Phosphorylated pp125FAK content was markedly decreased in hippocampal slices subjected to oxygenglucose deprivation. Thiopental and isoflurane significantly attenuated this phenomenon, possibly via PKC activation.
Keywords: anaesthetics volatile, isoflurane ; brain, hippocampal slice ; complications, aoxia-aglycaemia ; model ; rat
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Introduction |
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Materials and methods |
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Experimental protocol
Hippocampal slices (300 µm thickness) were incubated with 1 ml Ca2+-free artificial cerebrospinal fluid (CSF, 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 mixture. Ca2+ was omitted from the CSF to avoid tyrosine kinase activation at this stage of the experiment. Slices were incubated for 60 min at 37°C with moderate agitation under a humidified atmosphere of oxygen/carbon dioxide 95%/5% (vol/vol). In experiments with the NMDA challenge, MgCl2 was removed from the medium and replaced by CaCl2 (final concentration: 1.93 mM). Tetrodotoxin (1 µM) was added at the beginning of slice incubation to avoid indirect effects as a result of neuronal firing. Slices were transferred to air tight chambers (1 cm3 volume, 10 slices per chamber) and superfused at 10 ml min1 (during 10 and 30 min) with either the same oxygenated CSF (control) or a glucose-free CSF bubbled with nitrogen 95%carbon dioxide 5% containing 1 mM dithionite, an oxygen absorbent (glucose oxygen deprivation). ,
, and pH in the anoxic-aglycaemic solution were 2 (2) mm Hg, 40 (3) mm Hg and 7.4, respectively. Temperature in the chambers was servocontrolled to 37°C. After the desired period of simulated ischaemia, slices were recovered in oxygenated buffered CSF containing 4 mM glucose for 3 h to allow the development of neuronal death that may not be obvious immediately after ischaemic injury. The effects of thiopental (1, 10, and 100 µM, Specia Rhône Poulenc Rorer, Paris, France) and isoflurane (0.5, 1, and 2%, Abbott, Rungis, France) on pp125FAK under physiologic conditions and their sensitivity to three structurally distinct PKC inhibitors (chelerythrin, bisindolylmaleimide I and IX, 10 µM) and the NMDA receptor antagonist MK801 (10 µM), was studied first. Slices subjected to anoxia-aglycaemia were incubated either with or without the same isoflurane or thiopental, concentrations, respectively. Anaesthetics were present during the whole period of oxygenglucose deprivation. Isoflurane was delivered through the gas mixture used to bubble the chambers containing the slices via a calibrated vaporizer after a 30-min period of equilibrium at the appropriate concentration. Aqueous concentrations in the chambers were checked by gas chromatography.
Measurement of tyrosine-phosphorylated pp125FAK content
Phosphorylation of pp125FAK was measured by immunoblotting with both antiphosphotyrosine and specific anti-pp125FAK antibodies. At the end of reperfusion, slices were frozen in liquid nitrogen and homogenized by sonication in 200 µl of a solution of 1% (wt/vol) sodium dodecyl sulphate, 1 mM sodium orthovanadate and anti-proteases (50 µg ml1 leupeptin, 10 µg ml1 aprotinin, and 5 µg ml1 pepstatin) in water at 100°C. Homogenates were stored at 80°C until processing. The remaining slices were fixed in 4% formalin for 7 days, and embedded in paraffin for dead cell count. Equal amounts of protein (30 µg) were subjected to 6% (wt/vol) polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and transferred electrophoretically to nitrocellulose. Immunoblot analysis was performed with affinity-purified rabbit anti-phosphotyrosine antibodies SL2. Primary antibodies were labeled with peroxidase-coupled antibodies against rabbit IgG detected by exposure of autoradiographic films to a chemiluminescent reagent (ECL, Amersham, Little Chalfont, UK). Identification of phosphorylated pp125FAK was performed with a rabbit anti-Y397 FAK phosphospecific antibody (Biosource International, diluted 1:1000) after pooling five to eight independent samples. Immunoreactive bands were quantified using a computer assisted densitometer and expressed as a phosphotyrosine (pp125FAK, respectively) to ß-actin (quantified by using the specific monoclonal antiactin A5316 antibody (Sigma)) ratio (Cohu High Performance CCD camera, Gel Analyst 3.01 pci, Paris, France).
Quantification of cell death
Quantification of cell death was performed in the CA1 subfield area by fluorescent chromatin staining with application of 10 µg ml1 bis-benzimide (Hoechst 33258; Sigma) to fixed cells over 10 min. Serial 5-µm coronal sections were cut along the entire hippocampus. Stained cells were examined using a fluorescence microscope equipped with an appropriate filter (UV-2A; Zeiss, Oberkochen, Germany; excitation, 370 nm; emission, >400 nm) by an observer unaware of treatment assigned. Nuclei with features suggestive of neuronal death (pycnosis, i.e. condensation or fragmentation of chromatin) were counted in the CA1 area. The ratio of the number of pycnotic nuclei over the total number of stained nuclei was calculated.
Data analysis
Phosphorylation data (mean (SD)) were analysed using Fisher exact test and ANOVA with Scheffé's post-hoc correction for multiple comparisons and number of dead cells by the 2 test (Statistica 6.0 software). Data are expressed as a percentage of control tyrosine (pp125FAK, respectively) phosphorylation. P<0.05 was considered the threshold for significance.
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Results |
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Discussion |
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Methodological considerations
Since co-migrating bands could theoretically interfere with quantification of pp125FAK within the 125 kDa band identified with the non-specific phosphotyrosine antibody, a crucial methodological point was to ensure that the 125 kDa band identified by the non-specific anti-phosphotyrosine antibody corresponded to pp125FAK. For this purpose, we could have immunoprecipitated with specific anti-pp125FAK antibody before immunoblotting for phosphotyrosine, as was performed in previous studies originating from part of the same group,5 7 and demonstrated the co-migration of the phosphotyrosine containing band with pp125FAK by western blotting of a duplicate blot. We chose an alternative approach that consisted of using the anti-Y397 FAK phospho-specific antibody to quantify pp125FAK phosphorylation specifically induced by anaesthetics and pharmacologic agents. The specificity of this antibody for the phosphorylated form of pp125FAK has been demonstrated previously.5 The parallelism in phosphorylation intensity of the phosphotyrosine 125 kDa band and pp125FAK observed at clinically relevant concentrations of anaesthetics supports that the 125 kDa band phosphorylated by anaesthetics on the phosphotyrosine immunoblotting indeed corresponds to pp125FAK. Energy deprivation in hippocampal slices results in a cascade of events triggered by excitotoxic glutamate and leading to neuronal death by necrosis and/or apotosis.8 Unlike organotypic slice cultures, our model only allowed analysis of early events in this cascade.2 We observed that significant cell death was present after 30 (but not 10) min of ischaemia. Neuronal death was found after 10 min of ischaemia in the CA1 area in a previous study using a very similar approach.1 This difference may be explained by the use of a high superfusion rate (10 ml min1) in our study, which likely contributed to a decrease in the amount of excitotoxic glutamate present in the preparation and delayed the occurrence of cell death by necrosis.
Effects of oxygen glucose deprivation and anaesthetics on pp125FAK phosphorylation
Anoxia-aglycaemia produced a marked decrease in the content of phosphorylated pp125FAK at 30 min. This was likely to result from energy deprivation caused by ischaemia, as the phosphorylation process is ATP-dependent.8 9 The mechanisms involved in the attenuation by anaesthetics of the decrease in phosphorylated pp125FAK content by oxygenglucose deprivation (reduction in ATP depletion,9 reduction in glutamate release,1 2 effects on intracellular Ca2+ or on generation of reactive oxygen species) remain to be delineated. Under physiologic conditions, clinically relevant concentrations of thiopental and isoflurane significantly increased pp125FAK phosphorylation in a dose-dependent fashion. We also observed that the preservation of phosphorylated pp125FAK content by anaesthetics in the ischaemic slices was concentration-related. The block of pp125FAK phosphorylation by three structurally distinct PKC antagonists10 together with the lack of sensitivity to MK801 under physiologic conditions suggest that this effect is mediated directly or indirectly by PKC stimulation rather than NMDA receptor activation. However, these conclusions may not apply to the ischaemic conditions, as we did not examine the effects of PKC inhibitors and MK801 under oxygenglucose deprivation.
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Conclusion |
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Acknowledgments |
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References |
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2 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: 18995[CrossRef][ISI][Medline]
3 Girault JA, Costa A, Derkinderen P, Studler JM, Toutant M. FAK and PYK2/CAKß in the nervous system: a link between neuronal activity, plasticity and survival? Trends Neurosci 1999; 22: 25763[CrossRef][ISI][Medline]
4 Sonoda Y, Watanabe S, Matsumoto Y, Aizu-Yokota E, Kasahara T. FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathway in the hydrogen peroxide-induced apoptosis of the human ganglioblastoma cell line. J Biol Chem 1999; 274: 1056670
5 Derkinderen P, Siciliano J, Toutant M, Girault JA. Differential regulation of FAK+ and PYK2/CAK ß, two related tyrosine kinases, in rat hippocampal slices: effects of LPA, carbachol, depolarization and hyperosmolarity. Eur J Neurosci 1998; 10: 166775[CrossRef][ISI][Medline]
6 Dahmani S, Reynaud C, Keita H, Rouelle D, Mantz J. Anesthetic agents enhance tyrosine phosphorylation via activation of protein kinase C in the rat hippocampus. Anesthesiology 2001; 95 (Suppl): A694 (abstract)[CrossRef]
7 Siciliano JC, Toutant M, Derkinderen P, Sasaki T, Girault JA. Differential regulation of proline-rich tyrosine kinase 2/cell adhesion kinase ß (PYK2/CAK ß) and pp125FAK by glutamate and depolarization in rat hippocampus. J Biol Chem 1996; 271: 289426
8 Larsen M, Haugstad TS, Berg-Johnson J, Langmoen IA. The effects of isoflurane on brain amino acid release and tissue content induced by energy deprivation. J Neurosurg Anesthesiol 1998; 10: 16670[ISI][Medline]
9 Kass IS, Amorim P, Chambers G, Austin D, Cottrell JE. The effect of isoflurane on biochemical changes during electrophysiological recovery after anoxia in rat hippocampal slices. J Neurosurg Anesthesiol 1997; 9: 2896
10 Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000; 351: 95105[CrossRef][ISI][Medline]
11 Blanck JJ, Haile M, Xu F, et al. Isoflurane pre-treatment ameliorates postischemic neurologic dysfunction and preserves hippocampal Ca2+/Calmodulin-dependent protein kinase in a canine cardiac arrest model. Anesthesiology 2000; 93: 128593[CrossRef][ISI][Medline]
12 Sonoda Y, Kashara T, Yokota-Aizu E, Ueno M, Watanabe S. FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-AKt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line. J Biol Chem 1999; 274: 1056670
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