Microarray analyses of genes regulated by isoflurane anesthesia in vivo: a novel approach to identifying potential preconditioning mechanisms.

BACKGROUND: Although general anesthetics are recognized for their potential to render patients unconscious during surgery, exposure can also lead to long-term outcomes of both cellular damage and protection. As regards the latter, delayed anesthetic preconditioning is an evolutionarily conserved physiological response that has the potential for protecting against ischemic injury in a number of tissues. Although it is known that delayed preconditioning requires de novo protein synthesis, knowledge of anesthetic-regulated genes is incomplete. In this study, we used the conserved nature of preconditioning to analyze differentially regulated genes in 3 different rat tissues. We hypothesized that by selecting those genes regulated in multiple tissues, we could develop a focused list of gene candidates potentially involved in delayed anesthetic preconditioning. METHODS: Young adult male Sprague-Dawley rats were anesthetized with a 2% isoflurane/98% air mixture for 90 minutes. Immediately after anesthetic exposure, animals were euthanized and liver, kidney, and heart were removed and total RNA was isolated. Differential gene expression was determined using rat oligonucleotide gene arrays. Array data were analyzed to select for genes that were significantly regulated in multiple tissues. RESULTS: All 3 tissues showed differentially regulated genes in response to a clinically relevant exposure to isoflurane. Analysis of coordinately regulated genes yielded a focused list of 34 potential gene candidates with a range of ontologies including regulation of inflammation, modulation of apoptosis, regulation of ion gradients, and maintenance of energy pathways. CONCLUSIONS: Through using an analysis approach focusing on coordinately regulated genes, we were able to generate a focused list of interesting gene candidates with potential to enable future preconditioning studies.

Role for metallothioneins-I/II in isoflurane preconditioning of primary murine neuronal cultures.

BACKGROUND: Pretreatment with inhaled anesthetics, including isoflurane, can induce long-lasting cellular protection against ischemia-derived toxicity in multiple tissues, including brain tissue. Metal-regulatory genes, metallothioneins-I/II (MT-I/II), have been shown to protect against oxidative damage in multiple tissues. Furthermore, MT have been found to be differentially regulated in response to isoflurane and ischemic preconditioning. In this study, we assess the role of MT-I/II in mediating isoflurane preconditioning in primary neuronal-glial cultures. METHODS: Primary mouse neuronal-glial cultures were preconditioned with isoflurane (3 h, 1.5%) 24-96 h before 3-h oxygen-glucose deprivation (OGD, ischemic model). After OGD, isoflurane protection and responsiveness of MT-I/II knockdown and knockout cultures to preconditioning were assessed by lactate dehydrogenase release. Immunoassays for microtubule associated protein 2 and glial fibrillary acidic protein determined neuronal-glial sensitivity to preconditioning. MT-I/II messenger RNA was assessed by quantitative reverse transcriptase-polymerase chain reaction. Cultures transfected with exogenous MT-I/II were analyzed for protection against OGD toxicity. RESULTS: Isoflurane preconditioning reduced OGD-mediated toxicity by 11.6 +/- 7.9% at 24 h, with protection increasing to 37.5 +/- 2.5% at 72 h after preconditioning. Immunolabeling showed that neurons were more sensitive to OGD and more responsive to isoflurane preconditioning compared to glia. Quantitative reverse transcriptase-polymerase chain reaction showed MT-I/II messenger RNA were upregulated (approximately 2.5-fold) by isoflurane treatments. Also MT-I/II protein transfection significantly decreased OGD-mediated toxicity. Finally, knockdown and knockout of MT-I/II diminished and abolished isoflurane-mediated protection, respectively. CONCLUSIONS: MT-I/II play an important role in isoflurane-mediated delayed preconditioning against OGD toxicity of neuronal and glial cells in vitro.

Assessment of general anaesthetic cytotoxicity in murine cortical neurones in dissociated culture.

General anaesthetics are proposed to cause unconsciousness by modulating neuronal excitability in the mammalian brain through mechanisms that include enhancement of inhibitory GABA(A) receptor currents and suppression of excitatory glutamate receptor responses. Both intravenous and volatile agents may produce neurotoxic effects during early postnatal rodent brain development through similar mechanisms. In the following study, we investigated anaesthetic cytotoxicity in primary cortical neurones and glia from postnatal day 2-8 mice. Cultures at 4-20 days in vitro were exposed to combinations of ketamine (100 µM to 3 mM), nitrous oxide (75%, v/v) and/or isoflurane (1.5-5%, v/v) for 6-12 h. Neuronal survival and cell death were measured via microtubule associated protein 2 immunoassay and lactate dehydrogenase release assays, respectively. Clinically relevant anaesthetic concentrations of ketamine, nitrous oxide and isoflurane had no significant neurotoxic effects individually or when given as anaesthetic cocktails, even with up to 12 h exposure. This lack of neurotoxicity was observed regardless of whether cultures were prepared from postnatal day 0-2 or day 8 mice, and was also unaffected by number of days in vitro (DIV 4-20). Significant neurotoxic effects were only observed at supraclinical concentrations (e.g. 1-3 mM ketamine). Our study suggests that neurotoxicity previously reported in vivo is not due to direct cytotoxicity of anaesthetic agents, but results from other impacts of the anaesthetised state during early brain development.