During a certain phase in brain development, called the brain growth spurt, neurons seem to be particularly susceptible to neurodegenerative influences. In humans, this sensitive period ranges from the third trimester of pregnancy to the age of about 2 years. The rodent brain goes through a comparable developmental period in the first 3 weeks after birth [1–3]. Anaesthetics have a major influence on brain physiology. Neuroprotective influences on the adult brain have been demonstrated for anaesthetic agents such as propofol, barbiturates and volatile anaesthetics . Interestingly, Ikonomidou et al.  reported in 2000 that N-methyl-D-aspartate (NMDA)-glutamate-receptor antagonists and γ-aminobutyric acid type A-receptor agonists (GABAA-RA) can induce neurodegeneration in the embryonic brain. Until now, the neurodegenerative effects of various anaesthetic agents and sedatives such as ketamine, propofol, midazolam, isoflurane and nitrous oxide have been shown in the neonatal rodent brain [5,6]. For those anaesthetics, as well as for sevoflurane, a mechanism of action via GABAA agonism or glutamate antagonism or both is known [5,7,8].
Premature infants, newborns and pregnant women may undergo anaesthesia and sedation during various diagnostic and surgical procedures. Some anaesthetics used in paediatric anaesthesia have not yet been examined concerning their neurodegenerative effects. Sevoflurane is the main induction agent in paediatric anaesthesia and a widely used substance for the maintenance of general anaesthesia. For mask induction, it displays favourable properties such as a sweet smell and fast onset and offset . Despite its widespread use in paediatric anaesthesia, sevoflurane has not yet been analysed with regard to possible neurodegenerative effects on developing neurons. Therefore, this study was designed to investigate the time and concentration dependence of its effects on primary neuronal cultures of the rat.
Primary cortical neuronal culture
Primary neuronal cultures were prepared as described elsewhere . Briefly, Wistar rat embryos (E16-E18) (Forschungseinrichtung für Experimentelle Medizin, Charité-Universitätsmedizin Berlin, Germany) were used for cell culture preparation [10,11]. All procedures were performed according to the local guidelines for animal research approved by the Charité-Universitätsmedizin Berlin. Cerebral cortices were dissected in serum-free neurobasal medium with B27 supplement (Gibco, Invitrogen GmbH, Karlsruhe, Germany) and the meninges were removed. After mild trypsinization [15 min at 37°C in trypsin/ethylenediaminetetraacetic acid (0.05/0.02%)] in phosphate-buffered saline (PBS), the fragments were rinsed twice with PBS and once with dissociation medium (neurobasal medium with 2% B27 supplement, 1% penicillin and streptomycin and 1% L-glutamine) and subjected to mechanical dissociation by repeated aspirations through a Pasteur pipette in dissociation medium. Cell suspension was pelleted by mild centrifugation (1200 U min−1, 2 min at room temperature) and seeded in flat-bottom wells in neurobasal medium with B27 supplement containing 2 mmol l−1 L-glutamine, at a density of 3 × 106 ml−1. Prior to cell plating, wells were treated with poly-D-lysine (0.5% w/v in water; Sigma, St Louis, Missouri, USA) for 1 h at room temperature and rinsed twice with water. Cells were kept in a fully humidified incubator at 37°C under 5% CO2. After 4 days in vitro (DIV), 50% of the medium was removed and replaced with fresh medium. Cultures obtained under these conditions appeared to contain mostly neuronal cells (99%) determined by indirect immunocytochemistry for neurofilament.
Cell treatment with sevoflurane
After a total of 14–16 DIV, cells were exposed in two series to either 4 or 8 Vol.% sevoflurane (Abbott, Wiesbaden, Germany) via a vaporizer (Sevorane, Draeger-Vapor 2000, Draeger, Germany) for 3, 6, 9, 12, 24 and 48 h. Control cells remained untreated and measurements were taken at the same time points as the sevoflurane-treated cells.
Therefore, cell cultures were placed in the incubation chamber (Billups-Rothenburg, Del Mar, California, USA) and were then perfused with fresh air (21% O2, 5% CO2 and 69% N2) containing 4 or 8 Vol.% sevoflurane [12,13] After 10 min with a flow rate of 6 l min−1, the resultant atmosphere of the chamber reached a steady state level of 4 or 8 Vol.% sevoflurane measured by a Capnomac gas monitor (Datex-Ohmeda, Helsinki, Finland). The sevoflurane concentration did not change within the incubation time of 24 or 48 h. Control cells were superfused for the same length of time with fresh air only. Subsequently, the chambers were sealed and placed in the same incubator.
In order to explore the influence of GABAA receptors, untreated cell plates were preincubated with the GABAA receptor antagonists gabazine (0.1 mmol l−1) or picrotoxin (0.1 mmol l−1) for 30 min. After preincubation, these cultures were exposed to 8 Vol.% sevoflurane in the presence of the antagonists. Cell viability tests were also performed and assessed after 3, 6, 9, 12, 24 and 48 h of incubation and were compared with the controls, which were exposed to sevoflurane only.
To evaluate the mechanisms by which sevoflurane affects neuronal cells, cultures were additionally treated with the pan-caspase inhibitor zVAD-fmk [0.04 mmol l−1; Promega, Mannheim, Germany] and inhibitors of extracellular signal-regulated kinase [0.01 mmol l−1; (ERK1/2)-U0126, Cell Signaling Technology, Danvers, Massachusetts, USA] or phosphatidylinositol-3-kinase [0.01 mmol l−1; (PI3K)-ly294002, Cell Signaling Technology] in combination with sevoflurane 8 Vol.%.
Cell viability assay
The methyltetrazolium (MTT) assay is based on the reduction of the tetrazolium salt (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide) (Sigma) to coloured formazan by active dehydrogenases within live mitochondria to determine cell viability . Neuronal cells were plated onto 96-well plates coated with poly-D-lysine (Sigma) in 100 μl aliquots (60 000 cells per well) and treated with sevoflurane as described. Control cells were kept in neurobasal medium. After 3, 6, 9, 12, 24 and 48 h, 10 μl MTT at a final concentration of 0.5 mg ml−1 was added for 2 h. Insoluble purple formazan grains were solubilized by adding 100 μl of 10% sodium dodecyl sulphate – 0.01 N HCl. Absorbance at 570 nmol l−1 and a reference wavelength of 630 nm were analysed using a microplate reader (BioRad, Munich, Germany).
Apoptosis and necrosis detection ELISA
To quantify apoptosis and necrosis, histone-complexed DNA fragments, from the cytoplasm of apoptotic cells or when released from necrotic cells, were detected immunochemically and the amount was determined by spectrophotometry. After incubation of cells with sevoflurane as described, 20 μl of cell-incubating medium containing DNA from necrotic cells that leaked through the membrane during incubation was transferred to a streptavidin-coated well. The remaining medium was discarded. To determine the amount of apoptotic nucleosomes, apoptotic cells were lysed, centrifuged and an aliquot of the supernatant was likewise transferred to a streptavidin-coated well. The oligosomal DNA was quantitated using Cell Death Detection ELISAPlus (Roche Diagnostics, Mannheim, Germany, Cat No. 11 920685 001), according to the manufacturer's protocol. Results are expressed as absorbance from the supernatant of the lysate as well as from the incubating medium of the sevoflurane-treated cells against the values of untreated cells as controls.
Light phase contrast photomicrographs
To visualize the cell damage caused by sevoflurane (8 Vol.%), we took representative photomicrographs of exposed cultures and control cultures with an Olympus digital camera (Olympus, Hamburg, Germany).
Statistical significance was evaluated with the one-way analysis of variance (ANOVA) and with Bonferroni's multiple comparison test. The software used was GraphPad prism 4.03 (GraphPad software, Inc., San Diego, California, USA). Statistical significance was defined as a P value less than 0.05.
Cell viability for different sevoflurane concentrations
To determine the influence of sevoflurane alone in different concentrations on neuronal survival, the cell viability was evaluated. After incubation of the cultured cells with 4 and 8 Vol.% sevoflurane for 3, 6, 9, 12, 24 and 48 h, cell viability was measured by using MTT reduction (Fig. 1). The test series with 4 Vol.% sevoflurane showed no significantly reduced cell viability until 12 h of exposure. After 24 h, cell viability was reduced significantly (48.6 ± 9.9%, P < 0.001) compared with the untreated control group. The cell cultures treated with 8 Vol.% sevoflurane displayed no significant decrease in cell viability in the first 6 h. In these test series, cell viability was reduced significantly after 9 h (66.4 ± 10.9%, P < 0.01). Prolonged exposure of cultures to sevoflurane led to a more pronounced cell death in both groups. Until 9 h of exposure to sevoflurane, cell viability values were significantly lower in cultures treated with 8 Vol.% sevoflurane compared with cells with the same duration of exposure incubated with 4 Vol.%. From 12 h of exposure, no significant differences could be shown between both groups (Fig. 1).
Light phase contrast photographs taken to illustrate the microscopic differences showed a marked effect on cells after incubation with 8 Vol.% sevoflurane for 24 h (Fig. 2).
Cell viability for sevoflurane with GABAA-receptor antagonists
After 3, 6, 9, 12, 24 and 48 h, no significant differences in cell viability of cells treated with 8 Vol.% sevoflurane and the GABAA-receptor antagonists gabazine or picrotoxin compared with control cells (sevoflurane, 8 Vol.%) could be demonstrated (Fig. 3).
Evaluation of type of cell death
To further specify the type of cell death, we analysed the cell viability values after coincubation of cell cultures for 24 h with 8 Vol.% sevoflurane and inhibitors of the apoptotic pathway (Fig. 4). Incubating the cells with zVAD-fmk (pan-caspase inhibitor) did not significantly increase the number of surviving cells. ERK1/2 and PI3K are mediators of the antiapoptotic pathway. To evaluate their influence on the antiapoptotic pathway, cells were exposed to U0126 (inhibitor of ERK1/2) and ly294002 (inhibitor of PI3K). This experiment did not show any additional loss in cell viability. Furthermore, evaluation of the type of cell death by using Cell Death Detection ELISA revealed no statistically significant differences.
The present study was performed to investigate time-dependent and concentration-dependent effects of sevoflurane on developing neurons. We, therefore, used primary neuronal cultures of embryonic rats. Furthermore, the involvement of its main mechanism of action on the described effects should be examined. The main finding of the present study is that sevoflurane has no effect on neuronal cell viability in clinically relevant concentrations and exposure times. Neuronal damage was only observed after exposure times and concentrations that are relatively high in comparison with its use in clinical practice. GABAA-receptor antagonists did not abrogate sevoflurane-induced cell damage.
Neurodegenerative influences of anaesthetic agents for the developing brain are the focus of various publications. Most studies published in this field presented data on the neurodegenerative effects of ketamine, an NMDA-receptor-blocking anaesthetic agent that is not often used any more in paediatric anaesthesia . Extended neurodegeneration after application of ketamine and other NMDA-receptor antagonists was shown in the brain of neonatal rats  and mice . A recent study performed by Fredriksson et al. interestingly shows that the coadministration of GABAergic substances like barbiturates and propofol potentiates neonatal brain cell death caused by ketamine. Furthermore, ketamine at nonapoptotic concentrations can impair dendritic arbor development of GABAergic neurons as shown for cultured rat neurons . Another substance that acts mainly via the inhibition of the NMDA receptors, nitrous oxide , showed the induction of behavioural changes as well as neurodegenerative effects in different in-vivo settings [19,20].
Neurodegenerative effects have also been demonstrated for substances with predominantly GABAA-agonistic mechanisms of action. The studies performed on neonatal mice [2,16,21] showed neurodegeneration following application of benzodiazepines, propofol and barbiturates. Additionally, those studies [2,16,21] showed much more intense neurodegeneration triggered by combinations of anaesthetics such as thiopental and propofol, ketamine and propofol, midazolam and ketamine, or diazepam and phenobarbital. In contrast to glial cells, GABAergic neurons displayed vulnerability to propofol administration in rat glial-cell and neuronal-cell cocultures . Additional data obtained from cell culture experiments showed that propofol in concentrations not sufficient to induce cell death could, in contrast to midazolam, alter dendritic differentiation .
Our data extend findings made for the volatile anaesthetic isoflurane [12,24]. Wise-Faberowski et al.  observed neurodegenerative effects in organotypic hippocampal slice cultures after only 5 h of exposure to 1.5 Vol.% isoflurane and Jevtovic-Tedrodovic et al.  analysed the effects of isoflurane alone or in combination with midazolam and nitrous oxide after just 6 h in an in-vivo model of 7-day-old rats. We were able to demonstrate neurodegeneration for the volatile anaesthetic sevoflurane, particularly used in paediatric anaesthesia. Our data indicate a clear concentration and time dependence of the onset of significant neurotoxic effects. Namely, exposure to clinically relevant concentrations of sevoflurane (4 Vol.%) induced significant neurodegeneration only after 24 h. Using high concentrations (8 Vol.%), we could determine the onset of neurodegenerative effects after just 9 h.
The concentration of 8 Vol.% sevoflurane exceeds commonly used minimal alveolar concentrations (MACs) of ca. 3.3 Vol.% in human neonates [25,26] and of 3.74 Vol.% in 9-day-old rats . Even though concentrations of up to 8 Vol.% are used in the inspiratory line of the respirator , these concentrations are only applied for a matter of minutes during the induction of anaesthesia and are not measured in the expiratory line after reaching a steady state. When using the 4 Vol.% concentration, which is close to clinically relevant concentrations, we were able to determine a significant decrease in cell viability only after 24 h of exposure. Thus, sevoflurane displays no neurodegenerative effects in the model used when applied in concentrations and exposure times relevant in paediatric anaesthesia. As ours is an in-vitro model, we could not exclude the possibility that irreversible neuronal damage could occur in vivo at sevoflurane concentrations and exposure times much lower than those that cause cell death in vitro. In additional experiments, we evaluated cell viability following a 3 h-long sevoflurane exposure and 21 h washout (data not shown). Cell viability was reduced at a concentration of 8 Vol.% but not at 4 Vol.%. This model – widely used in studies for detecting neuroprotection after isoflurane preconditioning followed by ischaemia in brain slice cultures [13,29] – could be the basis for further investigations before stating that sevoflurane does not affect neuronal viability or whether sevoflurane could induce delayed apoptotic response. In-vivo studies [3,30] found that there is a time window from embryonic day 19 to postnatal day 14 when developing neurons are particularly sensitive to anaesthetics. Within this period, the brain of postnatal day 7 rats is thought to be particularly sensitive to neurotoxic effects . Neuronal maturation in primary cultures was initiated between 7 and 11 DIV . We used mature cultures at 14–16 DIV for our experiments. Considering that immature neuronal cultures are more sensitive, corresponding experiments should be done with immature cultures 5 DIV.
As we know, the GABAA receptor is the main receptor on which sevoflurane and other volatile anaesthetics act [7,8]. Hyperpolarization of immature neurons by GABA agonists may be a possible mechanism of the dose-dependent and time-dependent toxicity of sevoflurane . Even though sevoflurane enhances inhibitory postsynaptic channel activity at the GABAA receptors, picrotoxine and gabazine, as GABAA-receptor antagonists , could not reduce neuronal damage.
In diverse models of neurodegeneration by anaesthetic substances, apoptosis was defined as the underlying mechanism for injury. Exposure to ketamine, midazolam or a combination of these drugs triggered apoptotic neurodegeneration in the developing mouse brain . Apoptosis measured with Fluoro-Jade staining was increased in brains of mice treated with ketamine and propofol or ketamine and thiopental or a high dose of propofol alone . In our study, sevoflurane showed neuronal damage predominantly via necrosis. Inhibition of the extrinsic pathway of apoptosis, via activation of caspase-3 with zVAD-fmk, could not avoid the degeneration of sevoflurane-treated cells . On the other hand, we know that there is an intrinsic pathway in apoptosis. Inhibition of this pathway mediated by ERK1/2 and PI3K increased apoptosis in a model of hypoxia-induced apoptosis in neonatal brain slices . In cultures of immature oligodendrocytes hyperoxia induced apoptosis and reduced levels of phosphorylated ERK1/2 and PI3K. Oestrogen antagonized this effect and significantly reduced apoptosis . Activation of PI3K or ERK1/2 protects against apoptosis [37,38]. In our experiments, inhibition of the antiapoptotic pathway showed no additional sensitivity to apoptosis. Even though the intrinsic and extrinsic apoptotic pathways were not activated in our model, it has been reported that both pathways are activated in vivo by isoflurane in neonatal rats . Other investigators used detection of caspase-3 activation, either by western blot or by immunohistochemistry, to determine apoptotic cell death in the neonatal brain [40,41].
Even though this study is limited as an in-vitro model with neuronal cells, we demonstrated for the first time that, at concentrations and exposure times relevant for clinical anaesthesia, sevoflurane displays no neurodegenerative effects. This indicates the possibility of developing in-vivo models to describe time slots for safe anaesthesia with concentrations as low as possible, while still capable of maintaining a sufficient anaesthetic plane. Further studies should be done to investigate delayed effects of sevoflurane and to identify the underlying mechanisms of neuronal damage during sevoflurane anaesthesia. Direct in-vitro comparison studies would be important in establishing a difference between volatile anaesthetics. In addition, future clinical studies will be necessary to develop safe anaesthetic protocols.
The present work was supported by internal university research grants of the Charité-Universitätsmedizin Berlin. We thank Anne Carney for editorial assistance.
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