1 Department of Anesthesiology, Tokyo Metropolitan Fuchu Hospital, 2-9-2 Musashidai, Fuchu-shi, Tokyo 183-0042, Japan. 2 Department of Anesthesiology and Critical Care Medicine, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
* Corresponding author. E-mail: tkaneko{at}fuchu-hp.fuchu.tokyo.jp
Accepted for publication June 27, 2005.
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Abstract |
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Methods. Primary cultures of rat cortical neurones were exposed to non-lethal oxygenglucose deprivation (OGD), i.e. ischaemic preconditioning, for 30 min, 100 µM of diazoxide, a potent opener of the mitochondrial KATP (mitoKATP) channels, for 60 min or 1.4% isoflurane for 3 h. Lethal OGD was performed for 120 min 24 h after preconditioning stimuli. Neuronal injury was assessed by measurement of lactate dehydrogenase (LDH) efflux into the medium 24 h after lethal OGD, and neural viability was determined by proliferation assay. Gene and protein expression was confirmed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analysis 24 h after preconditioning stimuli.
Results. All preconditioning stimuli resulted in a significant decrease in LDH activity and maintained neuronal viability. These effects were abolished by 5-hydroxydecanoate, a selective inhibitor of the mitoKATP channel. Quantitative RT-PCR and Western blot analysis demonstrated that there was no significant difference between Kir6.2 mRNA and protein levels. All preconditioning stimuli resulted in 2-fold increases in EAAC1 mRNA and protein compared with control.
Conclusions. Isoflurane induced late preconditioning in cultured rat cortical neurones. Ischaemic and pharmacological preconditioning with diazoxide and isoflurane induced ischaemic tolerance in the cultured neurones via mitoKATP channels without an increase in Kir6.2 expression, and induced upregulation of EAAC1 expression.
Keywords: anaesthetics volatile, isoflurane ; ions, ion channels ; model, rat, cortical neurones ; model, preconditioning ; pharmacology, glutamate
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Introduction |
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Administration of isoflurane at clinically relevant concentrations profoundly alters the genetic programme in the myocardium, comparable with brief ischaemic episodes.4 This change in gene expression pattern directly reflects the pharmacological power of volatile anaesthetics. It has been reported that anaesthetic preconditioning (APC) with isoflurane produces dose-dependent neuroprotection via activation of KATP channels after focal cerebral ischaemia in rats,5 and that preconditioning brain slices with isoflurane 15 min before oxygenglucose deprivation (OGD) reduces Purkinje cell injury and death via modulation of glutamate transporter activity.6 Because the genetic profile of preconditioning is characterized by transcriptional changes of numerous genes involved in ion channel and membrane transport,7 APC with isoflurane may modulate expression of Kir6.2 (the ion-conducting subunit of the metabolically responsive KATP channel) and EAAC1 (neuronal glutamate transporter).
Therefore we tested the hypothesis that exposure to isoflurane 24 h before OGD induces late preconditioning in cultured rat cortical neurones. In addition, we tested the hypothesis that preconditioning elicits changes in Kir6.2 and EAAC1 expression.
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Methods |
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Cell culture
Primary cultures of cortical neurones from embryonic day 1618 Wistar rats were used as previously described.8 Briefly, foetuses were decapitated and cortical tissue was collected under sterile conditions. Meninges were removed and cortical tissue was dissociated in L-cysteine 5 mM (Sigma, USA), papain 10 U ml1 (ICN, USA) and DNase I 0.01% (Sigma, USA) at 37°C. Dissociated neurones were washed in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, USA), centrifuged and gently resuspended in neurone-defined serum-free Neurobasal medium (Gibco-BRL, USA) supplemented with B-27 (Gibco-BRL, USA), L-glutamine 0.5 mM (Gibco-BRL, USA) and gentamycin 2 µg ml1. The cells were plated at 8.5 x 105 cells ml1onto 100-mm culture dishes coated with poly-L-lysine 500 µg ml1 (Sigma, USA). The culture dishes were stored in humidified 95% air5% carbon dioxide at 37°C. Cytosine arabinoside 10 µM was added after 24 h to prevent the growth of non-neuronal cells. The medium was changed twice weekly and experiments were performed on day in vitro (DIV) 8. At this time, no more than 5% of the cells exhibited immunoreactivity to the antibody directed against glial fibrillary acidic protein.
Preconditioning and OGD
Cortical cultures were subjected to OGD injury using protocols described previously.9 Cultures were placed in an anaerobic incubator (BL-40 M, Juji Field, Japan), washed three times and replaced with glucose-free balanced salt solution (BSS) (NaCl 116 mM, KCl 5.4 mM, MgSO4 0.8 mM, NaH2PO4 1.0 mM, NaHCO3 26.2 mM, CaCl2 1.8 mM, glycine 0.02 mM and phenol red 2 mg l1) flushed with 5% carbon dioxide85% nitrogen10% hydrogen. Cell cultures were incubated in this solution at 37°C for a designated period to produce either non-lethal (30 min) or lethal (120 min) OGD. The oxygen concentration in the culture medium was monitored with an oxygen meter (DO-21P, DKK-TOA, Japan). OGD was terminated by removing the dishes from the anaerobic incubator, returning the stored medium to the dishes and returning the dishes to a standard cell incubator containing normal atmospheric oxygen5% carbon dioxide at 37°C. Control cell cultures not deprived of oxygen and glucose were exposed to BSS containing 6 mM glucose but were not bubbled with anaerobic gas, and were stored in the standard incubator.
Cultures were preconditioned based on a previously published method7 10 11 (Fig. 1).
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Quantitation of neuronal injury and viability
Neuronal injury was assessed by measurement of LDH efflux into the bathing medium 24 h after lethal OGD. In neuronal cultures, LDH activity in the medium correlated with the number of damaged cells.13 LDH activity was measured using an LDH Cytotoxicity Detection Kit (Takara, Japan). The culture medium was sampled and centrifuged to remove cellular debris from the supernatant. Subsequently, LDH reagent (lactate, NAD+, diapholase, 2-[4-indophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride) was added to 100 µl of the sample. The reaction was stopped by 0.2 N HCl after 30 min at room temperature and the absorbance at 490 nm was recorded. Analysis was performed on a fluorescence spectrophotometer (UV-1600, Shimadzu, Japan). The small amount of LDH present in the medium of sister cultures without lethal OGD was subtracted from the levels measured in the experimental conditions to yield the LDH signal specific to experimental injury. Data were expressed as a percentage of total LDH that was determined for each experiment by assaying the supernatant of sister cultures after 24 h of exposure to Triton X-100 1%. The data were obtained in three independent experiments with at least eight wells per condition.
Neuronal viability was quantified using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, USA) 24 h after lethal OGD. The assay reagent, which contained a novel tetrazolium compound and an electron coupling reagent, was added to the culture medium and the cultures were incubated 1 h at 37°C in a humidified atmosphere containing 5% carbon dioxide. The quantity of formazan produced as measured by the amount of 490 nm absorbance on a fluorescence spectrophotometer was directly proportional to the number of living cells in culture.14 The viability of each sample was calculated as a percentage of the absorbance of the sample cultured without lethal OGD. The data were obtained in three independent experiments with at least eight wells per condition.
Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
The mRNA (derived from 1 x 107 cells) was isolated from rat primary neurones using a QuickPrep micro mRNA Purification Kit (Amersham Biosciences, USA) 24 h after preconditioning. The mRNA concentration was determined by spectrophotometry at 260 nm. The first-strand cDNA was synthesized in 20-µl reaction solutions from 500 ng of mRNA using reverse transcriptase with oligo(dT) primer (Invitrogen, USA). The PCR primers were designed on the basis of the published mRNA sequences. To avoid amplification of the genomic DNA, each pair of primers was derived from the coding regions intervened with introns. The primers had the following sequences. For the 640 base product for ß actin: sense, 5'-TGCCCATCTATGAGGGTTACG-3'; antisense, 5'-TAGAAGCATTTGCGGTGCACG-3'. For the 386 base product for Kir6.2: sense, 5'-CTGCCTTCCTTTTCTCCATC-3'; antisense, 5'-TTACCACCCACACCGTTCTC-3'. For the 476 base product for EAAC1: sense, 5'-TCAATGCGTTGAGTGACGC-3'; antisense, 5'-GCTGATCGTGATGATCTGCC-3'. B-Actin was used as a standard for constitutive expression. All PCRs were performed in a final volume of 20 µl containing dNTP (0.2 mM), Taq polymerase (1 U), the primers (1 µM), and cDNA (0.5 µl) using a thermal cycler (PC707, ASTECH, Japan) under the following conditions: 94°C for 3 min; 3035 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 40 s; final extension at 72°C for 10 min.15 All products were assayed in the linear response range of the RT-PCR amplification process. The PCR products were separated by 3% Nusieve agarose gel electrophoresis and analysed by Electrophoresis Documentation and Analysis System 290 (Kodak, Japan) after ethidium staining.
Western blot analysis
Cell homogenates were prepared by homogenizing the cells 24 h after preconditioning in 50 mM TrisHCl pH 7.4 containing EDTA 1 mM, EGTA 1 mM, and protease inhibitor cocktail (Complete Mini EDTA-free, Roche Molecular Biochemicals, Germany). The crude homogenates were centrifuged at 1000g for 10 min at 4°C. The protein concentrations in the supernatants were determined by the bicinchoninic acid method with bovine serum albumin (BSA) as a standard using a BCA Protein Assay Reagent Kit (Pierce, USA). Equivalent amounts (100 µg) of protein samples were separated by 10% (Kir6.2) and 8% (EAAC1) sodium dodecylsulphatepolyacrylamide gels, electrotransferred onto nitrocellulose membranes (Bio-Rad, USA) as previously described,11 16 and then blocked with 5% skimmed milk in phosphate-buffered saline containing 0.1% Tween-20 at room temperature for 1 h. These membranes were probed with specific antibodies to Kir6.2 (1:2000) or EAAC1 (1:500), and all blots were also probed with ß-actin antibody (1:5000) to control for errors in protein sample loading and transferring during western blot analysis. Membranes were developed using affinity-purified peroxidase-labelled secondary antibodies (1:5000) and visualized on film with ECL Western Blotting Detection Reagents (Amersham Biosciences, USA) according to the manufacturer's directions. Immunoreactivity was quantified using Scion Image Beta 4.02 Win software (Scion, USA). In some cases, different exposures of the film were used to quantify different immunoreactive bands to ensure that the signal was within the linear range.
Statistics
All quantitative data were expressed as mean (SD). Multiple comparisons among groups were performed by one-way analysis of variance (ANOVA), followed by post hoc BonferroniDunn tests. P-values <0.05 were considered to be statistically significant.
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Results |
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Discussion |
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Initial events in preconditioning induction may involve opening the KATP channels via the activation of adenosine A1 receptors.12 The activation of KATP channels hyperpolarizes the neuronal cell membrane, thereby protecting the neurone from detrimental depolarization. Interestingly, the mitoKATP channel now appears to feature prominently in both phases of preconditioning. It is conceivable that administration of volatile anaesthetics, including isoflurane, which also activate KATP channels may produce late preconditioning. It has been reported that 1 h exposure to isoflurane 1.52.25% for 5 days induced late preconditioning via mitoKATP channels.5 Our study has demonstrated that opening of mitoKATP channels induced late-phase IPC and PPC with diazoxide and isoflurane, because administration of 5-HD before OGD blocked the preconditioning effects. On the other hand, our hypothesis that preconditioning would elicit changes in Kir6.2 gene expression was not confirmed. The identity of the mediator through which isoflurane exerts this effect and the signal transduction pathway leading to activation of mitoKATP channels are unknown. Capin and colleagues19 have speculated that reactive oxygen species (ROS) may mediate isoflurane-induced preconditioning, because ROS can mediate IPC and PPC with diazoxide. It has also been reported that late preconditioning with isoflurane could occur via the signalling pathway involving protein kinase C (PKC) or mitogen-activated protein kinase (MAPK), leading to the activation of NOS with subsequent synthesis of NO and opening of the mitoKATP channel.20
Ischaemia alters the properties of the neuronal plasma membrane, and this change could suppress extracellular release of glutamate during the second period of ischaemia. Glutamate uptake via sodium-dependent high-affinity excitatory amino acid transporters (EAATs) is the primary mechanism for clearance of extracellular glutamate in the cerebral cortex and is essential for terminating the postsynaptic action of glutamate. During cerebral ischaemia the extracellular cerebral glutamate content increases to neurotoxic levels. Although some extracellular glutamate arises from presynaptic vesicular release, this process is ATP dependent and hence may not be the main source of extracellular glutamate under hypoxic conditions. In comparison, hypoxia disrupts the ionic gradient across the plasma membrane, leading to a rise in intracellular Na+, which binds to and induces reversal of the EAAT, thereby facilitating glutamate efflux into the extracellular space. EAAT reversal may play a major role in hypoxia-induced glutamate release. There are five different EAAT isoforms in the mammalian cerebrum, designated EAAT1 (GLAST), EAAT2 (GLT-1), EAAT3 (EAAC1), EAAT4 and EAAT5. GLAST and GLT-1 are primarily astrocytic, whereas EAAC1 is found predominantly in neurones. It has been reported that IPC diminishes the increase in extracellular glutamate caused by OGD and increases cellular glutamate uptake and expression of EAAC1 protein.21 In addition, 3 h exposure to isoflurane 2% increased the expression and activity of EAAC1 in cultured rat C6 glioma cells by stabilizing EAAC1 mRNA and proteins via PKC-independent pathways.16 Our current study has also shown that IPC and PPC with diazoxide and isoflurane induced upregulation of EAAC1 expression in cultured rat cortical neurones, and this upregulation of EAAC1 expression which increases extracellular glutamate uptake and prevents EAAT reversal may be essential for preconditioning. However, what is the relationship between upregulation of EAAC1 expression and the opening of mitoKATP channels? Heurteaux and colleagues22 have reported that KATP channel openers hyperpolarized glutamate-sensitive neurones, thus conferring resistance to the depolarization induced by the increase in intracellular Na+ and preventing EAAT reversal. The preconditioning effect of isoflurane observed in our study may be due to the prevention of EAAT reversal via activation of KATP channels. Further studies are needed to test these hypotheses.
Our study has several potential limitations. First, although neuronal KATP channels may be formed through physical association of the pore-forming Kir6.2 subunit with the regulatory sulfonylurea receptor SUR1, only Kir6.2 expression was investigated in our study. However, SUR1-based KATP channels are not essential for neuronal preconditioning or augmentation of neurodegeneration by 5-HD.23 Secondly, our preconditioning experiments were performed on DIV 8, because hypoxic sensitivity increases between DIV 6 and DIV 8 in cortical neurones.24 Cultured cortical neurones become less tolerant to hypoxic stress with further maturation. This is similar to what is seen in newborn and adult brain tissues. This is also true for cultured hippocampal neurones, which exhibit increased hypoxic susceptibility after DIV 7. Therefore it appears that a critical developmental transition in terms of neuronal vulnerability to stress occurs in cultured neurones after 7 days. A variety of factors are believed to contribute to this phenomenon, including membrane protein expression, metabolism and release of excitatory amino acids. Because glutamate receptor expression increases during development and sensitivity to glutamate excitotoxicity increases with neuronal maturation, the observed differences in hypoxic susceptibility between neuronal ages may be associated with the developmental increase in glutamate toxicity. Another possible drawback of our study is that 5-HD was used as a selective inhibitor of the mitoKATP channel. Although an important piece of evidence for implicating mitoKATP channels as mediators of preconditioning is the consistent inhibitory effect of 5-HD, Hanley and colleagues25 have recently shown that ß-oxidation of 5-HD or its metabolic intermediates may be responsible for the inhibitory effects of 5-HD on preconditioning of the heart. Thus 5-HD may no longer be considered a suitable pharmacological tool for the identification of mitoKATP channels. However, this study has not necessarily disproved the inhibitory effect of 5-HD on mitoKATP channels. Further studies are required to assess the inhibitory effect of 5-HD on preconditioning of the brain.
In conclusion, we have shown that isoflurane induces late preconditioning in cultured rat cortical neurones. We have also demonstrated that IPC and PPC with diazoxide and isoflurane induced ischaemic tolerance via mitoKATP channels without an increase in Kir6.2 expression, and induced upregulation of EAAC1 expression in cultured rat cortical neurones.
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