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BJA Advance Access published online on September 2, 2005
British Journal of Anaesthesia, doi:10.1093/bja/aei228
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© The Board of Management and Trustees of the British Journal of Anaesthesia 2005. All rights reserved. For Permissions, please e-mail: journal.permissions@oxfordjournals.org
Accepted June 27, 2005

Laboratory Investigation

Late preconditioning with isoflurane in cultured rat cortical neurones

T. Kaneko 1*, K. Yokoyama 2, and K. Makita 2

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

* To whom correspondence should be addressed.
T. Kaneko, E-mail: tkaneko{at}fuchu-hp.fuchu.tokyo.jp


  Abstract

Background. We tested the hypothesis that isoflurane induces late preconditioning in cultured rat cortical neurones and preconditioning elicits changes in expression of Kir6.2 (the ion-conducting subunit of the metabolically responsive ATP-sensitive potassium (KATP) channel) and EAAC1 (neuronal glutamate transporter).

Methods. Primary cultures of rat cortical neurones were exposed to non-lethal oxygen-glucose 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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