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The Journal of Physiology logoLink to The Journal of Physiology
. 2012 Jun 6;590(Pt 16):4093–4107. doi: 10.1113/jphysiol.2012.233965

Metabotropic actions of the volatile anaesthetic sevoflurane increase protein kinase Mζ synthesis and induce immediate preconditioning protection of rat hippocampal slices

Jun Wang 1, Fanli Meng 1,2, James E Cottrell 1, Todd C Sacktor 2,3,4,5, Ira S Kass 1,2,3,5
PMCID: PMC3476650  PMID: 22674720

Abstract

Anaesthetic preconditioning occurs when a volatile anaesthetic, such as sevoflurane, is administered before a hypoxic or ischaemic insult; this has been shown to improve neuronal recovery after the insult. We found that sevoflurane-induced preconditioning in the rat hippocampal slice enhances the hypoxic hyperpolarization of CA1 pyramidal neurons, delays and attenuates their hypoxic depolarization, and increases the number of neurons that recover their resting and action potentials after hypoxia. These altered electrophysiological effects and the improved recovery corresponded with an increase in the amount of a constitutively active, atypical protein kinase C isoform found in brain, protein kinase M zeta (PKMζ). A selective inhibitor of this kinase, zeta inhibitory peptide (ZIP), blocked the increase in the total amount of PKMζ protein and the amount of the activated form of this kinase, phospho-PKMζ (p-PKMζ); it also blocked the altered electrophysiological effects and the improved recovery. We found that both cycloheximide, a general protein synthesis inhibitor, and rapamycin, a selective inhibitor of the mTOR pathway for regulating protein synthesis, blocked the increase in p-PKMζ, the electrophysiological changes, and the improved recovery due to sevoflurane-induced preconditioning. Glibenclamide, a KATP channel blocker, when present only during the hypoxia, prevented the enhanced hyperpolarization, the delayed and attenuated hypoxic depolarization, and the improved recovery following sevoflurane-induced preconditioning. To examine the function of persistent PKMζ and KATP channel activity after the preconditioning was established, we administered 4% sevoflurane for 30 min and then discontinued it for 30 min before 10 min of hypoxia. When either tolbutamide, a KATP channel blocker, or ZIP were administered at least 15 min after the washout of sevoflurane, there was little recovery compared with sevoflurane alone. Thus, continuous KATP channel and PKMζ activity are required to maintain preconditioning protection. We conclude that sevoflurane induces activation of the mTOR pathway, increasing the new protein synthesis of PKMζ, which is constitutively phosphorylated to its active form, leading to an increased KATP channel-induced hyperpolarizaton. This hyperpolarization delays and attenuates the hypoxic depolarization, improving the recovery of neurons following hypoxia. Thus, sevoflurane acts via a metabotropic pathway to improve recovery following hypoxia.

Key points

  • Volatile anaesthetics, such as sevoflurane, have been shown to reduce neuronal damage when administered as preconditioning protective agents before hypoxia or ischaemia.

  • Most rapid onset protective effects of anaesthetics have been thought to be due to direct effects on ion channels in the neurons and do not require the activation of biochemical pathways or protein synthesis

  • We found that sevoflurane activates the mammalian target of rapamycin (mTOR) biochemical pathway, increasing the rapid synthesis and activation of the protein kinase, PKMζ, a PKC isoform critical for maintaining long-term potentiation and long-term memory storage; this, in turn, increases the activity of KATP channels, and induces an increased hyperpolarization during hypoxia. This reduces and delays the hypoxic depolarization and improves neuronal recovery from hypoxia.

  • Thus, it would be advantageous to choose an anaesthetic, such as sevoflurane, that rapidly preconditions and protects neurons from hypoxia and ischaemia for surgeries in which the brain is at risk for damage.

Introduction

Cerebral hypoxia and ischaemia are important causes of death and disability, particularly during surgical procedures. Patients undergoing surgical procedures such as endarterectomies and cardiopulmonary bypass surgery have a much greater risk of stroke in the peri-operative period due to thrombus formation and/or microemboli (Shaw et al. 1985; Wolman et al. 1999; Aronow et al. 2010). Recent studies have found that short, non-damaging ischaemic episodes before a longer ischaemic episode prevent the damage that would normally occur after the longer ischaemia (Gidday, 2006; Malhotra et al. 2006; Roth et al. 2006). However, it is not possible to subject a compromised patient to a short non-damaging ischaemia in order to protect them from a longer damaging ischaemia; the compromised patient may have a lower threshold for permanent neuronal damage. There also may be minor and currently unrecognized deleterious effects of these short ischaemic periods (Tanay et al. 2006). Surgery requires anaesthesia; therefore, choosing an anaesthetic that induces cerebral preconditioning subjects the patient to no additional risk and indeed may provide protection against neuronal damage. Whereas other studies have examined delayed preconditioning that is expressed starting 12 h after the anaesthetic administration (Xiong et al. 2003; Zheng & Zuo, 2004; Bickler et al. 2005; Payne et al. 2005; Sanders et al. 2010), our studies have focused on immediate preconditioning using the volatile anaesthetic sevoflurane that protects rapidly after the treatment (Wang et al. 2007).

Anaesthetics have direct actions on ion channels, such as enhancing GABAA receptor activity, as well as more lasting metabotropic effects on biochemical signalling pathways (Wang et al. 2007; Alkire et al. 2008). These metabotropic effects can alter cell excitability as well as initiate long-term changes in neurons. Anaesthetic-induced preconditioning is probably a consequence of the activation of these biochemical signalling pathways because it persists after the anaesthetic is removed (Sanders et al. 2010).

Sevoflurane before hypoxia improves the recovery of resting and action potentials of CA1 pyramidal cells in rat hippocampal slices 60 min after hypoxia and the improvement was blocked by chelerythrine, a non-specific blocker of protein kinase C isozymes (Wang et al. 2007). The protection was associated with an enhanced hypoxic hyperpolarization and an attenuated hypoxic depolarization, which were also blocked by chelerythrine (Wang et al. 2007). In that study we also examined long-term neuronal damage to CA1 neurons after global cerebral ischaemia. We found that sevoflurane-induced immediate preconditioning increases the number of histologically intact neurons in the CA1 region 6 weeks after global cerebral ischaemia; this indicates that anaesthetic preconditioning can provide long-term protection against ischaemic neuronal damage (Wang et al. 2007). In the current paper we use a selective peptide inhibitor of PKMζ, zeta inhibitory peptide (ZIP), to block its activity (Ling et al. 2002). We measure the expression of PKMζ with sevoflurane-induced preconditioning and examine whether the mTOR pathway is implicated in this expression and the improved recovery.

Methods

Ethical approval

The experiments were approved by the Institutional Animal Care and Use Committee of the State University of New York, Downstate Medical Center and carried out in accordance with these guidelines, which conform to the principles of UK regulations. The number of animals in each experimental group is given in the figure legends; a total of 239 rats were used. The minimal number of animals to assure statistical reliability were used. Drug concentrations were determined from previous studies by others that examined the effect of LTP-induced changes in PKMζ (Ling et al. 2002; Cracco et al. 2005; Serrano et al. 2005; Pastalkova et al. 2006). Male Sprague–Dawley rats (100–120 days old) were anaesthetized with 2% isoflurane for 2 min in a Plexiglas chamber using a calibrated isoflurane vaporizer. Two minutes of 2% isoflurane anaesthesia did not alter recovery of the evoked responses in previous experiments (Wang et al. 2006). Adequate anaesthesia was confirmed by the loss of the righting reflex and the lack of any response to handling. The animal was then decapitated with a guillotine, and its brain was quickly removed and placed into chilled (2–4°C) artificial cerebrospinal fluid (aCSF). The hippocampus was rapidly removed from the brain and sliced.

Intracellular recording

Hippocampal slices 400 μm thick were sectioned in chilled aCSF (4–6°C) using a vibratome. The slices were stored in a beaker containing aCSF saturated with 95% O2 and 5% CO2, and remained there for approximately 2 h at 25°C. The composition of the aCSF was (in mmol l−1): NaCl, 126; KCl, 3; KH2PO4, 1.4; NaHCO3, 26; MgSO4, 1.3; CaCl2, 1.4 glucose, 4, at pH 7.4, and was equilibrated with 95% O2–5% CO2. Slices were transferred to a tissue chamber and maintained at 37°C in this chamber. Hypoxia was generated by switching the gas to 95% N2–5% CO2 (Wang & Kass, 1997; Wang et al. 1999).

The hippocampal slice was submerged in the recording chamber and perfused with aCSF at a rate of 3.0 ml min−1. A bipolar stimulating electrode was placed in the Schaffer collateral/commissural pathway and a CA1 pyramidal neuron was impaled with a glass micropipette filled with 4 m potassium acetate (70–120 MΩ). Only neurons with stable resting potentials of at least −60 mV for 15 min with high-amplitude, short-duration action potentials that showed spike frequency accommodation and were activated by short-latency Schaffer collateral/commissural stimulation were examined (Wang et al. 1999, 2006).

The untreated group received 25 min of perfusion, then 10 min of hypoxia followed by 60 min of reperfusion. The sevoflurane-treated groups received 5 min of perfusion, 15 min of 4% sevoflurane, 5 min washout and then 10 min of hypoxia (no anaesthetic) followed by 60 min of reperfusion. ZIP (5 μm; Anaspec, San Jose, CA, USA) was added 10 min before and during sevoflurane pretreatment and washed out 5 min before hypoxia. Dizocilpine (MK-801 25 and 100 μM), a non-competitive NMDA receptor antagonist, was added 10 min before and during sevoflurane pretreatment and cycloheximide (200 μm) or rapamycin (200 nM) were added 15 min before and during sevoflurane pretreatment; all drugs were washed out 5 min before hypoxia. Glibenclamide (5 μm) or tolbutamide (5 μm) were present only during the hypoxia.

For Western blot analysis, the samples were removed at the indicated time points and the CA1 regions were dissected under a microscope on powdered dry ice. The region analysed contained tissue from the stratum radiatum to the alveus including the CA1 pyramidal cell layer (Kass & Lipton, 1989). The tissue was homogenized with 15 strokes of a Teflon–glass Potter–Elvehjem tissue grinder at 4°C in 300 μl of buffer (50 mm Tris–HCl (pH 7.5), 1 mm EDTA, 1 mm EGTA, 5 mm 2-mercaptoethanol, 0.1 mm phenylmethylsulfonyl fluoride, aprotinin (17 kallikrein units ml−1), 5 mm benzamidine and 0.1 mm leupeptin), centrifuged at 3000 g for 5 min, and the supernatant solution was used as the total protein (Libien et al. 2005). Protein concentrations were determined by the bicinchoninic acid method (BCA, Pierce). The samples were boiled in SDS–polyacrylamide gel sample buffer (50 mm Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1%β-mercaptoethanol, 12.5 mm EDTA, 0.02 % bromophenol blue) at 95°C for 10 min, and then 10 μg total protein from each sample was loaded onto adjacent lanes of 8% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels. Following electrophoresis, the proteins were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), and blocked for 90 min in 1% bovine serum albumin and 1% haemoglobin in Tris-HCl, pH 7.5 (10 mm), NaCl (150 mm) and non-ionic detergent (0.2%) (TBSN) (reagents were from Sigma, St Louis, MO, USA, unless otherwise stated). The nitrocellulose membrane was incubated in primary antisera overnight at 4°C. The membrane was washed in TBSN and then incubated for 90 min with secondary antibody coupled to alkaline phosphatase (1:2000) (anti-mouse IgG-alkaline phosphatase, Sigma Cat. no. 9316; anti-rabbit IgG-alkaline phosphatase, Sigma Cat. no. 3687). The blots were developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) (KPL Inc., Gaithersburg, MA, USA), scanned and analysed with NIH Image. PKC isozymes were assayed by Western blots with optimized concentrations of phosphorylated PKMζ T560 antiserum (phospho PKCzeta pT560 rabbit antibody; Epitomics Inc. Cat. no. 2200-1; 1:100). All gels had a non-hypoxic control group and the density of the PKMζ band in each experiment was normalized to the density of PKMζ in this non-hypoxic untreated control, which was set to 1. The levels of PKMζ in fractions from the hippocampal slices were compared by loading equal amounts of total protein from the fractions on each lane of the gel. The densities of the protein bands on the Western blot membrane were in the linear range of detection as determined with NIH Image software after membranes were scanned with a high-resolution flatbed scanner. The house-keeping protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH mouse monoclonal antibody; Chemicon International, Cat. no. MAB374) was used as an internal loading control.

Statistics

For the intracellular recording experiments, a chi-square test, an ANOVA followed by the Newman–Keuls multiple comparison tests, or Student's t test was used to test significance (Prism 4, GraphPad Software, San Diego, CA, USA). Any stated difference in the Results section is a statistically significant difference. One-way analysis of variance (ANOVA) followed by a post hoc Student–Neuman–Keul test was used to analyse the significant difference among different experimental groups. All the data are expressed as mean ± SEM. The difference between different groups was considered significant if P < 0.05.

Results

Effect of sevoflurane and PKMζ inhibition on electrophysiological responses during and after hypoxia

The membrane potential of CA1 pyramidal cells from untreated rat hippocampal slices was −64 mV. During hypoxia these neurons initially hyperpolarize by 3 mV. This is followed by a slow depolarization and then a rapid and complete depolarization to −0.4 mV. When 4% sevoflurane is applied for 15 min and then washed out 5 min before a 10 min hypoxic interval, the amplitude of the initial hyperpolarization is enhanced (from 3.1 to 6.2 mV), the latency before the rapid hypoxic depolarization is prolonged (from 240 to 485 s) and the membrane potential at 10 min of hypoxia is less depolarized (from −0.4 to −36 mV) (Fig. 1). Sevoflurane-mediated immediate preconditioning significantly increased the number of neurons that recovered their resting potentials 60 min after hypoxia compared with the untreated hypoxic group (from 0 to 88%).

Figure 1. Effect of zeta inhibitory peptide (ZIP) and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia.

Figure 1

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A, resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia; in untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane is given via a calibrated vaporizer in the gas stream for 15 min and then washed out for 5 min before hypoxia, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. The figure shows the potential for 15 min after hypoxia; the data in the text and the statistics are from 60 min after the hypoxia. When 5 μm ZIP is applied 10 min before and during the sevoflurane treatment, the effects of sevoflurane on the electrophysiological changes are blocked. All points are the mean ± SEM (n = 8 slices and animals for each group, one cell was recorded from each slice). When a bar is not shown the size of the standard error is less than the size of the symbol for that point. B, a continuous recording from a single CA1 pyramidal cell from each group before, during and after hypoxia; the data in A come from 10 such recordings from each group. The 3 responses after hypoxia in the sevoflurane-alone hypoxia group are due to Schaffer collateral/commissural stimulation and do not represent spontaneous activity. The horizontal calibration is indicated by the 10 min hypoxia bar, the vertical calibration is ∼60 mV from before hypoxia to after hypoxia in the untreated and ZIP + sevo-treated groups (the scale is the same for the sevoflurane preconditioning group).

To assess the role of PKMζ, slices were treated with ZIP (5 μm) 10 min before and during the sevoflurane preconditioning. Treatment with ZIP attenuated the enhanced hyperpolarization induced by sevoflurane (6.2 vs. 1.4 mV) and the neurons depolarized completely by the end of the hypoxic period. ZIP reduced the number of neurons that recovered their resting potentials 60 min after hypoxia with sevoflurane-induced preconditioning (88 vs. 0%). Thus, ZIP blocked the physiological and protective effects of sevoflurane preconditioning. The same concentration of scrambled sequence ZIP (5 μm) did not block the protective preconditioning effects of sevoflurane (Table 1). It is possible that hypoxia even without sevoflurane may enhance PKMζ synthesis since the hypoxic hyperpolarization with ZIP and sevoflurane preconditioning was lower than that in untreated hypoxia (1.4 vs. 3.1 mV; Table 1). The increase in PKMζ with sevoflurane was next tested by Western blot analysis.

Table 1.

Effects of ZIP on electrophysiological changes induced by sevoflurane preconditioning

Groups Amplitude of initial hyperpolarization Latency of rapid deporlarization Membrane potential at 10 min hypoxia Recovery (%) n
Untreated 3.1 ± 0.3 240 ± 11 −0.4 ± 0.2 0 10
Sevoflurane, 4% 6.2 ± 0.4* 485 ± 24* −36 ± 4.5* 88* 8
ZIP, 1 μm+ Sevo, 4% 5.6 ± 0.5* 462 ± 22* −32 ± 4.0* 83* 6
ZIP, 5 μm+ Sevo, 4% 1.4 ± 0.2*† 336 ± 13*† −2.5 ± 1.1† 0† 6
ZIP Scrambled, 5 μm+ Sevo, 4% 5.8 ± 0.4* 465 ± 14* −34 ± 4.4* 100* 6
Sevo, 4%+ tolbutamide, 5 μm 1.1 ± 0.2*† 310 ± 15*† −1.6 ± 0.5† 0† 5
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*

P < 0.05, compared with untreated group;

†

P < 0.05, compared with Sevo 4% group.

Effect of sevoflurane and a PKMζ inhibitor on the expression of PKMζ and phosphorylated PKMζ before, during and after hypoxia

Total PKMζ protein was measured on a Western blot in untreated and sevoflurane-treated tissue, before hypoxia, at 10 min of hypoxia and at 60 min after hypoxia (Fig. 2A and B). When sevoflurane (4%) was present for 15 min and then washed out for 5 min there were significantly increased PKMζ levels compared with the non-treated group (1.6 vs. 1). The PKMζ inhibitor ZIP (5 μm) attenuated the increase of PKMζ protein expression with sevoflurane (1.1 vs. 1.6), such that the PKMζ level between the non-hypoxic untreated group and the sevoflurane plus ZIP-treated group was not significantly different. PKMζ levels were also measured at the end of 10 min of hypoxia. Hypoxia alone increased these levels compared with the non-hypoxic control (1.4 vs. 1). Sevoflurane preconditioning increased this further (2.2 vs. 1.4); however, the level with sevoflurane plus ZIP was less than that with sevoflurane alone during hypoxia. Thus, 10 min of hypoxia stimulated the expression of PKMζ, and sevoflurane pretreatment increased the level of the kinase over hypoxia alone and sevoflurane alone. The sevoflurane-pretreated neurons recovered their physiological activity after the end of hypoxia; we therefore measured PKMζ levels 60 min after the end of hypoxia. Sevoflurane preconditioning not only enhanced PKMζ levels before and during hypoxia, but the enhancement was maintained for at least 60 min after the end of hypoxia. Neither the untreated nor the sevoflurane treated with ZIP slices showed enhanced PKMζ levels 60 min following hypoxia.

Figure 2. Effect of ZIP and sevoflurane-induced preconditioning on protein kinase M zeta (PKMζ) protein levels before, during and after 10 min of hypoxia.

Figure 2

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Slices are either untreated, subjected to sevoflurane-induced preconditioning or ZIP 10 min before sevoflurane-induced preconditioning. A and C, For each Western blot an untreated non-hypoxic control is run on the same gel and used as a normalization standard; its density is set to 1 and all other bands are a ratio of this density. Six CA1 regions from tissue treated in the same chamber are from a single animal and are microdissected and pooled to run in a single lane. GAPDH is not affected by hypoxia and used to normalize the values for total protein within each lane. Each bar represents the mean and standard error of the mean from 10 animals. Sevoflurane-induced preconditioning significantly increased the amount of PKMζ protein before, during and after hypoxia. ZIP reduced the amount of PKMζ protein at these time points. * P < 0.05 compared with untreated group. B, representative Western blots from each experiment measuring PKMζ and GAPDH. C, effect of ZIP and sevoflurane-induced preconditioning on phosphorylated protein kinase M zeta (p-PKMζ) protein levels before, during and after 10 min of hypoxia. p-PKMζ is the activated form of PKMζ; unlike other members of the PKC family, PKMζ is constitutively phosphorylated and activated. Treatment is as described above. Sevoflurane-induced preconditioning significantly increased the amount of p-PKMζ protein before, during and after hypoxia. Zeta inhibitiory peptide reduced the amount of p-PKMζ protein at these time points. D, representative Western blots from each experiment measuring p-PKMζ and GAPDH.

PKMζ is active in its phosphorylated state, and under normal conditions it is rapidly phosphorylated after its synthesis and subsequently all of it is in the activated state. We measured the phosphorylated, activated PKMζ form to confirm this, especially during hypoxia, when the amount of ATP for phosphorylation could be limited (Fig. 2C and D). Sevoflurane significantly increased the level of phosphorylated PKMζ (p-PKMζ) before hypoxia, compared with the level in control untreated non-hypoxic tissue; ZIP blocked this increase. After 10 min of hypoxia and 60 min reoxygenation the increases in p-PKMζ in the sevoflurane pretreatment group were maintained and ZIP blocked the effects of sevoflurane. The untreated hypoxia group had a small but significant increase in p-PKMζ at 10 min hypoxia that was not sustained with reoxygenation. Thus, the results with the p-PKMζ mirrored those of PKMζ, indicating that most of total PKMζ was constituatively phosphorylated, similar to normal conditions in hippocampal slices. This suggests that the increase in p-PKMζ was due to new synthesis and not just phosphorylation of preformed PKMζ. The one exception to the parallel increases in PKMζ and p-PKMζ is during hypoxia, when there is a greater increase in non-phosporylated PKMζ; this may be due to the limited availability of ATP for phosphorylation during hypoxia.

Effect of protein synthesis inhibition on sevoflurane-induced preconditioning

In order to test whether de novo protein synthesis is required for sevoflurane-induced immediate preconditioning protection, we used the general protein synthesis inhibitor cycloheximide. Cycloheximide blocked sevoflurane-induced preconditioning protection. When cycloheximide (200 μm) was given 15 min before and during sevoflurane application, it reduced the number of neurons that recovered compared with sevoflurane alone (13 vs. 83%) (Fig. 3). It also reduced the hypoxic hyperpolarization (2.8 ± 0.6 vs. 4.8 ± 0.6 mV) and the latency of the rapid depolarization (240 ± 20 vs. 441 ± 36 s) compared with sevoflurane alone; the levels of these parameters returned to those in untreated hypoxic tissue (2.6 ± 0.4 mV and 221 ± 24 s). The membrane depolarization at 10 min of hypoxia in the cycloheximide plus sevoflurane group (−2.8 ± 0.8 mV) was also similar to that in the untreated hypoxia group (−0.7 ± 0.5 mV) and significantly more depolarized than the sevoflurane preconditioning group (−14.5 ± 5.4 mV). These results indicate that immediate sevoflurane preconditioning requires new protein synthesis. Although delayed preconditioning, which takes at least 12 h to be expressed, requires new protein synthesis (Gidday, 2006), it is a novel finding that immediate preconditioning that is expressed within 20 min also requires new protein synthesis.

Figure 3. Effect of cycloheximide or rapamycin and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia.

Figure 3

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Resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia. In untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane is given in the gas stream for 15 min and then washed out for 5 min before hypoxia, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. The figure shows the potential for 20 min after hypoxia; the data in the text and the statistics are from the data at 60 min after the hypoxia. When either cycloheximide (200 μm), a general inhibitor of protein synthesis, or rapamycin (200 nm), a specific inhibitor of the mTOR pathway, is applied 15 min before and during the sevoflurane treatment, the effects of sevoflurane on the electrophysiological changes are blocked. All points are the mean ± SEM. When a bar is not shown, the size of the standard error is less than the size of the symbol for that point. N is equal to the number of slices and animals for each group; one cell was recorded from each slice. The number of animals for the untreated group is n = 8; the sevoflurane-induced preconditioning group n = 6; the sevoflurane plus cycloheximide group n = 8; the sevoflurane plus rapamycin group n = 8.

Effect of mTOR pathway inhibition on sevoflurane-induced preconditioning

It is important to understand which protein synthetic pathways are activated by sevoflurane preconditioning. The mammalian target of rapamycin (mTOR) pathway is a mitogenic protein synthesis-activating pathway that is associated with the metabolic state of a cell. As this pathway has been implicated in LTP-induced PKMζ synthesis, we examined this pathway to determine if it was involved in activating protein synthesis in response to sevoflurane (Fig. 3). A selective blocker of this pathway, rapamycin (200 nm), applied 15 min before and during sevoflurane, blocked the sevoflurane preconditioning-induced protection. None of the neurons recovered after sevoflurane-treated slices were pretreated with rapamycin, compared with 83% with sevoflurane preconditioning alone. Rapamycin attenuated the sevoflurane-enhanced hypoxic hyperpolarization (2.4 ± 0.2 vs. 4.8 ± 0.6 mV) and the increase in the latency of the rapid depolarization (225 ± 22 vs. 441 ± 36 s) with sevoflurane-induced preconditioning. The membrane potentials at 10 min hypoxia with rapamycin and sevoflurane were similar to that in the untreated hypoxic tissue (−1.6 ± 0.2 vs. 0.7 ± 0.5 mV), but were more depolarized than in the sevoflurane preconditioning group (−14.5 ± 5.4 mV).

Effect of cycloheximide and rapamycin on PKMζ synthesis

As in the previous experiments, sevoflurane again increased the expression of p-PKMζ when present for 15 min before hypoxia. If either cycloheximide or rapamycin was present before and during the sevoflurane application, the increase in p-PKMζ was blocked. This indicated that the increase in p-PKMζ requires new protein synthesis and that this synthesis is stimulated by activation of the mTOR pathway (Fig. 4A and B).

Figure 4. Effect of cycloheximide or rapamycin and sevoflurane-induced preconditioning on phosphorylated protein kinase M zeta (p-PKMζ) protein levels before, during and after 10 min of hypoxia.

Figure 4

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A, slices are either untreated, subjected to sevoflurane-induced preconditioning or either cycloheximide (200 μm) or rapamycin (200 nM) 15 min before and during sevoflurane application. For each Western blot an untreated non-hypoxic control is run on the same gel and used as a normalization standard; its density is set to 1 and all other bands are a ratio of this density. GAPDH is used to normalize the values for total protein within each lane. Six CA1 regions from tissue treated in the same chamber are from a single animal and are microdissected and pooled to run in a single lane. Each bar represents the mean and SEM from 10 animals. Sevoflurane-induced preconditioning significantly increased the amount of p-PKMζ protein before, during and after hypoxia. When either cycloheximide or rapamycin was present before the sevoflurane the amount of p-PKMζ protein was reduced, compared with sevoflurane-induced preconditioning alone. * P < 0.05 compared with untreated group. B, representative Western blots from each experiment.

Effect of NMDA receptor blockade on sevoflurane-induced preconditioning

The activation of NMDA receptors triggers LTP and its accompanying increase in PKMζ. We therefore tested whether blockade of the NMDA receptor during sevoflurane application blocked sevoflurane-induced preconditioning. When 4% sevoflurane was applied in the presence of concentrations of MK 801 sufficient to block the NMDA receptor, there was still significantly improved recovery with sevoflurane-induced preconditioning (Table 2). Thus, it appears that sevoflurane is inducing preconditioning independent of activation of the NMDA receptor.

Table 2.

Effects of MK801 on electrophysiological changes induced by sevoflurane preconditioning

Groups Amplitude of initial hyperpolarization Latency of rapid depolarization Membrane potential at 10 min hypoxia Recovery (%) n
Untreated 2.4 ± 0.15 288 ± 7 −0.3 ± 0.2 0 6
Sevoflurane, 4% 4.2 ± 0.25* 516 ± 15* −52 ± 9* 83 6
MK801, 25 μm+ sevo, 4% 3.4 ± 0.15* 438 ± 39* −30 ± 13* 50 6
MK801, 100 μm+ sevo, 4% 3.5 ± 0.20* 481 ± 29* −48 ± 12* 75 4
MK801, 25 μm 2.8 ± 0.14 351 ± 21 −0.8 ± 0.5 0 4
MK801, 100 μm 2.9 ± 0.15 408 ± 180* −15 ± 2 0 2
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*

P < 0.01 compared with untreated group.

Effect of KATP channel blockade during hypoxia on sevoflurane-induced preconditioning

In order to test the role of the KATP channel in the expression of sevoflurane-induced protection, we applied the KATP channel blocker glibenclamide only during the hypoxia. Glibenclamide completely blocked the hypoxic hyperpolarization and the preconditioning protection with sevoflurane (Fig. 5). Sevoflurane preconditioning increased the number of neurons that recovered their resting and/or action potentials after hypoxia from 0% to 83%; if glibenclamide is present only during the hypoxia after sevoflurane preconditioning, then none of the neurons recovered their resting or action potentials (0%). Sevoflurane-induced preconditioning increased the hypoxic hyperpolarization from 2.4 ± 0.2 mV to 4.5 ± 0.3 mV; glibenclamide reduced the hypoxic hyperpolarization with sevoflurane-induced preconditioning to 0.6 mV ± 0.1 mV, a level significantly below that for the untreated hypoxic slice. In the untreated hypoxic slices, glibenclamide also blocked the hypoxic hyperpolarization (0.2 mV ± 0.1 mV); this is similar to what we found with ZIP and provides evidence that sevoflurane enhances the hypoxic hyperpolarization by causing an increase in PKMζ, which then enhances the KATP channel. Glibenclamide attenuated the increase in the latency of the rapid depolarization with sevoflurane preconditioning from 483 ± 30 to 272 ± 9 s and the final level of depolarization from −16.7 ± 3.2 to −0.6 ± 0.3 mV. Another KATP channel blocker, tolbutamide (5 μm), also blocked the protective effects of sevoflurane-induced preconditioning (Table 1). Thus, sevoflurane's preconditioning effect appears to be due to delaying and attenuating the hypoxic depolarization by enhancing the hypoxic hyperpolarization that occurs directly after the onset of hypoxia.

Figure 5. Effect of glibenclamide and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia.

Figure 5

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A, resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia; in untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane-induced preconditioning is given, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. When 5 μm glibenclamide, a blocker of the KATP channel, is applied only during the hypoxia, the effects of sevoflurane-induced preconditioning on the electrophysiological changes are blocked. All points are the mean ± SEM (n = 10 slices and animals for each group; one cell was recorded from each slice). When a bar is not shown, the size of the standard error is less than the size of the symbol for that point.

Persistent PKMζ activity mediates the Effect of sevoflurane on a delayed administration of hypoxia

In order to examine whether KATP channel activity is required to maintain the preconditioning protection after its establishment, we prolonged the sevoflurane washout period to 30 min. We were able to demonstrate that 4% sevoflurane significantly improved recovery after 10 min of hypoxia, even after a 30 min washout (83 vs. 0%) (Fig. 6). The protective effect of sevoflurane-induced preconditioning was reduced by 5 μm tolbutamide (11 vs. 83%) and is therefore also probably due, at least in part, to activation of KATP channels.

Figure 6. Effect of tolbutamide and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia.

Figure 6

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Resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia; in untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane is given in the gas stream for 30 min and then washed out for 30 min before hypoxia, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. When tolbutamide (5 μm) is applied during the hypoxia, only after sevoflurane washout for 30 min, the effects of sevoflurane on the electrophysiological changes are blocked. The figure shows the potential for 25 min after hypoxia; the data in the text and the statistics are from the data at 60 min after the hypoxia. All points are the mean ± SEM. When a bar is not shown, the size of the standard error is less than the size of the symbol for that point. N is equal to the number of slices and animals for each group; one cell was recorded from each slice. The number of animals for the untreated group is n = 7; the sevoflurane-induced preconditioning group n = 6; the sevoflurane plus tolbutamide group n = 6.

Importantly, we next examined whether PKMζ activity was needed for the maintenance of preconditioning protection; previous experiments had only examined it before and during sevoflurane application but not during hypoxia. Sevoflurane was administered for 30 min and washed out for 30 min before 10 min of hypoxia; ZIP (5 μm) was applied 15 min after sevoflurane wash out (i.e., 15 min before hypoxia). ZIP reduced the sevoflurane preconditioning protection (20 vs. 80%) and its associated electrophysiological correlates (Fig. 7); this experiment implicates the importance of continual PKMζ activity for the maintenance of preconditioning protection and not just for its initiation.

Figure 7. Effect of PKMζ inhibitory peptide (ZIP) and sevoflurane-induced preconditioning on electrophysiological responses during and after 10 min of hypoxia.

Figure 7

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Resting potentials are recorded from CA1 pyramidal cells before, during and after 10 min hypoxia; in untreated slices (no sevoflurane) there is no recovery of the resting potential after hypoxia. When 4% sevoflurane is given in the gas stream for 30 min and then washed out for 30 min before hypoxia, the slices show an enhanced hyperpolarization, a delayed and attenuated depolarization, and recovery of the resting potential after hypoxia. When ZIP (5 μm) is applied 15 min before and during the hypoxia, the effects of sevoflurane on the electrophysiological changes are blocked. The figure shows the potential for 25 min after hypoxia; the data in the text and the statistics are from the data at 60 min after the hypoxia. All points are the mean ± SEM. When a bar is not shown, the size of the standard error is less than the size of the symbol for that point. Each treatment group contained 10 slices from 10 different animals; one cell was recorded from each slice.

Discussion

Hypoxia and ischaemia are important causes of neuronal damage and neurological dysfunction following stroke, cardiac arrest, traumatic brain injury and surgical procedures that may lead to a temporary block of the blood supply to the brain. These events cause a reduction of energy availability in the cell and lead to a loss of essential functions including ion pumping and protein synthesis (Kass & Lipton, 1982; Lipton, 1999; Raley-Susman et al. 2001). This, in turn, can lead to cell depolarization, activation of damaging pathways and eventual cell death. A short ischaemic period before a longer ischaemic period, however, has been shown to make neurons resistant to damage from the longer period of ischaemia; this has been termed ischaemic preconditioning and is thought to involve the synthesis of new proteins (Schurr et al. 1986; Xu et al. 2002; Gidday, 2006; Malhotra et al. 2006; Roth et al. 2006). The problem with ischaemic preconditioning as a therapeutic tool is that it may also cause damage (Tanay et al. 2006). Therefore, there has been a search for drugs that reduce damage when applied before the longer ischaemia. Volatile anaesthetics, such as sevoflurane, have been shown to protect in heart tissue during ischaemia (Stowe & Kevin, 2004; Tanaka, 2004). This is called anaesthesia-induced (or sevoflurane-induced) preconditioning. Sevoflurane preconditioning also enhances the recovery after hypoxia of CA1 pyramidal cells in rat hippocampal slices (Wang et al. 2007). Moreover, in an in vivo global ischaemic model, sevoflurane preconditioning improved CA1 pyramidal cell survival 1 and 6 weeks after ischaemia (Wang et al. 2007). Thus, results found in vitro 1 h after hypoxia in the hippocampal slice model used in the current experiments translate to long-term neuronal recovery after immediate sevoflurane preconditioning in vivo. Others have found that sevoflurane enhances long-term survival of neurons in vivo by altering biochemical pathways normally induced by ischaemia (Pape et al. 2006). Additionally, it is likely that the mechanisms found in this paper for our in vitro studies are also applicable to the in vivo studies of sevoflurane immediate preconditioning described above.

Previously, we demonstrated that 4% sevoflurane when applied shortly before but not during hypoxia improved recovery of the resting and action potentials after hypoxia. This improved recovery was blocked by the protein kinase C inhibitor chelerythrine, indicating that sevoflurane's actions were mediated by a metabotrophic response to improve recovery (Wang et al. 2007). The minimal alveolar concentration (the concentration at which 50% of the animals are anaesthetized) for sevoflurane is approximately 2.1%; it is approximately half as potent as isoflurane, another commonly used volatile anaesthetic agent. Concentrations as high as 8% sevoflurane have been used for the induction of anaesthesia, but a more common level for the maintenance of anaesthesia is 3–4% (Ebert & Schmid, 2009). In previous studies we found that both 2 and 4% sevoflurane induced preconditioning protection, but 2% required 60 min to establish its effect (Wang et al. 2007); in the current studies we used 4% to study the mechanism because its shorter onset of preconditioning protection allowed us to carry out intracellular experiments that would have been more difficult over longer time periods.

Protein kinase M zeta is a unique and interesting kinase whose synthesis is associated with long-term potentiation (LTP) in hippocampal brain slices, and learning and memory in animals (Pastalkova et al. 2006; Yao et al. 2008; Sacktor, 2011). LTP, a prolonged enhancement in synaptic transmission, has been shown to depend on PKMζ because blocking PKMζ activity with PKMζ inhibitory peptide, ZIP, or chelerythrine (Ling et al. 2002) blocks the formation and reverses the potentiation (Serrano et al. 2005; Pastalkova et al. 2006). Moreover, when ZIP was infused into the hippocampus of animals that had learned a specific task, the animals could no longer do that task even after the ZIP was washed out. However, those animals could learn and remember a new task after the ZIP was removed, indicating that the animals still had the capacity to learn but the memory of the old task was lost (Pastalkova et al. 2006). Our data indicate that sevoflurane-induced preconditioning protection also requires the activation of PKMζ. As PKMζ is expressed exclusively in brain tissue, it represents a unique pathway for sevoflurane-induced preconditioning cerebral protection (Hernandez et al. 2003). The increase of PKMζ we find with sevoflurane-induced preconditioning is similar to the amount of increase found after LTP in the CA1 region of the hippocampal brain slice (Osten et al. 1996). Whereas our current results demonstrate that PKMζ is necessary for sevoflurane-induced preconditioning, it may not be sufficient as other metabolic pathways have been shown to be important for volatile anaesthetic-induced preconditioning (Zheng & Zuo, 2004; Bickler et al. 2005; Sanders et al. 2010).

We found that sevoflurane increased the amount of PKMζ and phospho-PKMζ in the CA1 region of hippocampal slices. This increase was measured before the hypoxia, even though it was only 20 min after its application. The selective blocker of PKMζ, ZIP, prevented the increase in PKMζ and phospho-PKMζ and blocked the protective effect of sevoflurane-induced preconditioning. This indicates that synthesis and activation of PKMζ is necessary for sevoflurane-induced preconditioning. It is unlikely that an increase in phosphorylation of preformed PKMζ accounts for the increase in phospho-PKMζ and it is likely that the increase in PKMζ and phospho-PKMζ is due to new protein synthesis. There are three main reasons for this conclusion: (1) almost all PKMζ is rapidly and constituatively phosphorylated under basal conditions in hippocampal slices (Kelly et al. 2007); (2) PKMζ has been shown to enhance its own synthesis and blocking its activity reduces PKMζ levels during LTP; and (3) we found an increased level of total PKMζ that paralleled the increase in phospho-PKMζ, similar to that observed with LTP. In order to confirm this deduction, we measured the level of phospho-PKMζ after sevoflurane in the presence of the protein synthesis inhibitor cycloheximide. We found that cycloheximide blocked the increase in phospho- PKMζ induced by sevoflurane and also blocked sevoflurane preconditioning protection.

The current study on anaesthesia-induced preconditioning examines the mechanisms by which sevoflurane, when present before, but not during hypoxia, improves neuronal recovery after the hypoxia. Our studies focus on immediate preconditioning in which the protection from ischaemia begins shortly after the treatment (Wang et al. 2007); other studies have examined delayed preconditioning which is expressed starting 12 h after the ischaemia (Xiong et al. 2003; Zheng & Zuo, 2004; Bickler et al. 2005; Payne et al. 2005; Sanders et al. 2010). Whereas previous studies have recognized the importance of protein synthesis and gene activation for delayed preconditioning, the importance of protein synthesis was not recognized for immediate preconditioning because of time constraints. Gene expression and mRNA formation in the nucleus and protein synthesis and transport to the axons and dendrites would take too long to allow immediate preconditioning within 1 h. However, recent experiments examining LTP in dendrites indicate new protein can be synthesized rapidly from mRNA located in the dendrites (Casadio et al. 1999; Ling et al. 2002; Hernandez et al. 2003; Muslimov et al. 2004; Cracco et al. 2005; Kelly et al. 2007; Yao et al. 2008; Sacktor, 2011). Thus, immediate preconditioning may indeed require rapid de novo protein synthesis and this has been demonstrated in the current study.

The mTOR pathway is rapidly activated during LTP and leads to new protein synthesis of PKMζ (Kelly et al. 2007); this mitogenic pathway has also been implicated in hypoxia and the signalling pathways induced by hypoxia (Arsham et al. 2003; Martin & Hall, 2005). The regulation of the mTOR pathway has been extensively studied (Hay & Sonenberg, 2004) and has been shown to be important for synaptic plasticity (Casadio et al. 1999; Tang et al. 2002; Kelleher et al. 2004; Cracco et al. 2005). Disregulation of this pathway has been implicated during neuronal damage (Hoeffer & Klann, 2010). We tested whether this mitogenic pathway is important for sevoflurane-induced preconditioning. Rapamycin, a specific blocker of this protein synthesis-activating pathway, blocked protection by sevoflurane-induced preconditioning and also blocked the formation and activation of PKMζ. Thus, it appears that sevoflurane activates the mTOR pathway, and this leads to the synthesis of PKMζ. It is currently unclear how sevoflurane activates these pathways. Our current data indicate that sevoflurane is not enhancing glutamate NMDA receptors and thereby Ca2+ influx to activate these pathways, as occurs during LTP. However, sevoflurane has been shown to increase cytosolic Ca2+ levels by enhancing the release of Ca2+ from the endoplasmic reticulum and it is possible that the increased cytosolic Ca2+ can activate the mTOR pathway; this will need to be examined in future experiments (Bickler et al. 2009; Wei & Xie, 2009; Zhao et al. 2010).

Our data with the KATP channel inhibitors glibenclamide and tolbutamide indicate that the KATP channel is downstream of PKMζ because applying these drugs during the hypoxia, after the sevoflurane has been washed out, prevents the protection with preconditioning. Sevoflurane increased the hypoxic hyperpolarization, and this hyperpolarization appears to be primarily due to the increase in KATP channels because blocking the KATP channel dramatically decreases the hypoxic hyperpolarization. Indeed, the hyperpolarization was reduced to below the level with hypoxia alone, indicating that even in the absence of sevoflurane preconditioning, this channel plays a role in the hypoxic response.

In order to confirm the effect of agents on the expression of preconditioning, we altered the protocol to include a 30 min instead of a 5 min washout; we also extended the sevoflurane application to 30 min. We showed that 30 min of 4% sevoflurane followed by a 30 min washout enhanced hypoxic hyperpolarization, delayed depolarization and improved recovery following hypoxia. Tolbutamide, the KATP channel blocker, or ZIP, the inhibitor of PKMζ, prevented these preconditioning-induced changes when the agents were applied after the expression of preconditioning had been established. Thus, the persistent increase of PKMζ activity and KATP channel conductance is required for the expression of sevoflurane-induced preconditioning.

Volatile anaesthetics are known to alter ionotropic neuronal transmission; they have been shown to activate GABAA and inhibit NMDA and AMPA glutamatergic transmission (Alkire et al. 2008). Previous investigators have examined the role of these pathways in protecting against hypoxic and ischaemic damage (Bickler et al. 2003; Head & Patel, 2007). Anesthetics, however, affect these pathways only when they are continually present, and this may not be possible during ischaemia, which limits the clinical efficacy of this mechanism of action of these agents. In contrast, the metabotropic actions of the volatile anaesthetic sevoflurane are prolonged and maintained even when the anaesthetic is no longer present. The current experiments, in part, provide evidence to support the following putative pathway: sevoflurane activates the mTOR pathway (blocking mTOR pathway with rapamycin prevents preconditioning), which in turn induces the synthesis of the constitutively active protein kinase PKMζ (blocking PKMζ synthesis and activation with cycloheximide, rapamycin or ZIP prevents preconditioning); this causes an enhanced activity or increased number of open KATP channels in the membrane (blocking the KATP channels with glibenclamide blocks the hypoxic hyperpolarization and the preconditioning protection); this hypoxic hyperpolarization delays the hypoxic/ischaemic depolarization and reduces damage (Fig. 8). The importance of delaying and attenuating the spread of the depolarization during hypoxia and ischaemia for reducing the core of a cortical lesion has been demonstrated in vivo (Higuchi et al. 2002; Kobayashi et al. 2007; Sasaki et al. 2009).

Figure 8. Potential mechanism of sevoflurane-induced preconditioning.

Figure 8

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The black arrows do not imply a direct action – there may be intermediate effects.

Thus, we conclude that sevoflurane-induced preconditioning activates the mTOR pathway, enhancing PKMζ synthesis and activation, which in turn increases the activity of KATP channels, and induces hyperpolarization during hypoxia and ischaemia. This pathway leads to improved recovery from hypoxia.

Acknowledgments

The authors gratefully acknowledge the financial support of University Physicians of Brooklyn, Brooklyn Anesthesia Research; State University of New York Downstate Medical Center, Dean's fund pilot project award to I.S.K.; and National Institutes of Health grants to T.C.S. (R37 MH057068, R01 MH53576)

Glossary

mTOR

mammalian target of rapamycin

PKMζ

protein kinase M zeta

p-PKMζ

phospho-PKMζ

ZIP

zeta inhibitory peptide

Author contributions

J.W.: conception and design of the experiments; collection, analysis and interpretation of data. F.M.: conception and design of the experiments; collection, analysis and interpretation of data; drafting the article and revising it critically for important intellectual content. J.W. and F.M. contributed equally to the conception and design of the experiments and the collection and analysis of the data. J.E.C.: conception and design of the experiments and revising it critically for important intellectual content. T.C.S.: conception and design of the experiments; interpretation of data; revising the article critically for important intellectual content. I.S.K.: conception and design of the experiments; analysis and interpretation of data; drafting the article and revising it critically for important intellectual content. All authors approved the final version.

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