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Molecular Evidence of Late Preconditioning After Sevoflurane Inhalation in Healthy Volunteers

Section Editor(s): Durieux, Marcel E.; Gin, TonyLucchinetti, Eliana PhD*; Aguirre, José MD*; Feng, Jianhua MD, PhD*; Zhu, Min PhD*; Suter, Marc MD*; Spahn, Donat R. MD*; Härter, Luc PhD; Zaugg, Michael MD*

doi: 10.1213/01.ane.0000278159.88636.aa
Anesthetic Pharmacology: Preclinical Pharmacology: Research Report
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BACKGROUND: Late preconditioning by volatile anesthetics evolves in response to transcriptional changes. We hypothesized that sevoflurane inhalation would modify the transcriptome in human blood and modulate the expression of adhesion molecules in white blood cells consistent with the occurrence of a late preconditioning phase.

METHODS: Five healthy male subjects inhaled sevoflurane at an end-tidal concentration of 0.5%–1.0% for 60 min. Venous blood samples were collected at baseline, after 15 and 60 min of inhalation, and 6, 24, 48, and 72 h thereafter and immediately processed for flow cytometry and mRNA extraction and hybridization to Affymetrix U133 Plus 2.0 microarrays. Data were analyzed using Significance Analysis of Microarray and Gene Set Enrichment Analysis and confirmed by real-time reverse transcription polymerase chain reaction. L-selectin (CD62L) and β2-integrin (CD11b) expression was determined on granulocytes and monocytes using flow cytometry.

RESULTS: Sevoflurane inhalation rapidly and markedly altered gene expression in white blood cells. Key transcripts potentially involved in late preconditioning or organ protection including paraoxonase, 12-lipoxygenase, heat shock protein 40, chemokine ligand 5, and phosphodiesterase 5A were regulated in response to sevoflurane. Sevoflurane further decreased transcripts involved in peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling and fatty acid oxidation. Reduced L-selectin (CD62L) expression on granulocytes accompanied with increased resistance to inflammatory activation was present at 24 to 48 h after sevoflurane exposure.

CONCLUSIONS: Sevoflurane at subanesthetic concentrations modifies blood transcriptome and decreases the expression of the proinflammatory L-selectin (CD62L), consistent with a “second window of protection” in humans.

IMPLICATIONS: The authors demonstrate that a short period of sevoflurane inhalation at subanesthetic concentrations (<1%) alters the blood transcriptome in healthy volunteers. Flow cytometry analysis further demonstrated reduced L-selectin expression on granulocytes accompanied with resistance to inflammatory stimulation 24 to 48 h after sevoflurane exposure, consistent with the occurrence of a second window of protection in humans.

From the *Institute of Anesthesiology; and †Department of Trauma Surgery, University Hospital Zurich, Switzerland.

Accepted for publication June 4, 2007.

Supported by the Swiss National Science Foundation, Berne, Switzerland (Grant No. 3200B0-103980/1 and No. 3200B0-116110/1), a grant from the Olga Mayenfisch Foundation, Zurich, Switzerland, a grant from the Swiss Society of Anesthesiology and Reanimation (SGAR), Berne, Switzerland, a grant from Abbott AG, Baar, Switzerland, and the Fifth Frontiers in Anesthesia Research Award from the International Anesthesia Research Society, Cleveland, Ohio.

Reprints will not be available from the author.

Address correspondence to Michael Zaugg, MD, Institute of Anesthesiology, E-HOF, University Hospital Zurich and, Zurich Center for Integrative Human Physiology ZIHP, University of Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland. Address e-mail to michael.zaugg@usz.ch.

Preconditioning is a biological process observed in multiple organs, whereby a transient stressful stimulus induces a protective state against a more prolonged, potentially lethal insult. It can be induced by brief ischemic episodes or by drugs such as volatile anesthetics (1,2). This protection has two phases: an early phase, immediately operating after the application of the preconditioning stimulus and lasting for 2–3 h, and a late phase, evident after 12–24 h but lasting for up to 3 days. Although early preconditioning is predominantly based on multiple, fast-acting intracellular phosphorylation signaling steps (3), the second window is a result of transient altered gene activity (4) and depends on novel protein expression (5). Volatile anesthetic-induced preconditioning was reported to be effective in various cell types, including cardiac myocytes (6,7), and endothelial and smooth muscle cells (8). In addition, several experimental studies provide evidence of a “second window of protection” elicited by volatile anesthetics in mouse (9), rat (10–12), and rabbit hearts (13–15). In contrast, one study with isoflurane was unable to show delayed cardioprotection in an in vivo dog model (16). However, it is unknown whether late preconditioning elicited by volatile anesthetics also occurs in humans.

Recruitment of inflammatory cells to sites of ischemic injury contributes significantly to organ dysfunction. Focal accumulation of leukocytes is mediated by the interaction of selectins with their endothelial counterligands, while firm attachment of leukocytes and transmigration requires activation of β2-integrin (CD11b) (17). Using a highly controlled human in vivo model of endothelial dysfunction, we have recently shown that sevoflurane inhalation at subanesthetic concentrations decreases activation of β2-integrin (CD11b) on granulocytes and monocytes after ischemia-reperfusion of the forearm in healthy volunteers, consistent with an early preconditioning of the endothelium (18). We next wanted to know whether sevoflurane inhalation would also regulate gene and protein expression in the blood in accordance with the occurrence of a late window of anesthetic-induced preconditioning in humans. Specifically, we hypothesized that sevoflurane inhalation would modulate blood transcripts potentially involved in late protection and reduce the expression of L-selectin (CD62L) and β2-integrin (CD11b) 24–48 h later in humans.

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METHODS

Study Subjects

In this study, five healthy male volunteers with Caucasian genetic background, aged 32 ± 5 (27–39) yr and with a body mass index of 23 ± 6 (21–28) kg/m2 gave informed signed consent. All participants were nonsmokers and refrained from caffeine and dark chocolate containing endothelium-protective flavonoids from 24 h before until 72 h after sevoflurane inhalation. The subjects fasted overnight, and were pretreated with a single dose of oral ranitidine (300 mg) the evening before sevoflurane inhalation. The research project was performed in accordance with the Declaration of Helsinki (2000), and was approved by the local ethics committee of the University Hospital Zurich, Switzerland.

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Study Protocol

Figure 1 shows the time course of the experiments. After inserting a cannula into a cubital vein and the administration of 500 mL Ringer solution, the subjects inhaled sevoflurane in 50% oxygen to achieve a subanesthetic end-tidal concentration of between 0.5% and 1.0% for 60 min. Sevoflurane was inhaled by the spontaneously breathing volunteers using a facemask connected to the common gas outlet of an anesthesia machine (Siemens Servo 900D ventilator, Siemens Life Support Systems, Sona, Sweden), as previously described (18). Blood samples were taken from the cubital vein (opposite side of blood pressure cuff) before gas inhalation (baseline), after 15 and 60 min of sevoflurane administration, and 6, 24, 48, and 72 h thereafter. One week after sevoflurane inhalation, separate experiments, conducted in a crossover mode with the same individuals, served to clarify whether inhalation of 50% oxygen without sevoflurane and administration of a single oral dose of ranitidine (300 mg) would affect gene expression. Monitoring consisted of intermittent noninvasive arterial blood pressure measurements, 5-lead electrocardiogram, end-tidal CO2, end-tidal sevoflurane concentrations (Hellige VICOM-SM SMU 612; PPG, Freiburg, Germany), and Bispectral Index (A2000 monitor® with three adhesive electrodes to the forehead, single channel: Fp1–Fpz, version 3.3; Aspect Medical Systems, Natick, Massachusetts) for depth of sedation.

Figure 1

Figure 1

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Transcriptional Profiling of Human Blood Samples

Total RNA was extracted from whole blood samples (collected in tripotassium-EDTA tubes) after centrifugation using RiboPure™-Blood Kit (Ambion, Huntington, United Kingdom). Microarray analysis was performed following the “minimum information about a microarray experiment” guidelines (19). Briefly, the quality of the isolated RNA was determined with a NanoDrop ND 1000 (NanoDrop Technologies, Delaware) (260/280 nm ratio between 1.8 and 2.1 and 28S/18S ratio within 1.5–2). Probe labeling and purification was achieved as previously described (20). From four of the five subjects, samples collected at baseline, 15 min, 1, 6, and 24 h were processed and hybridized to an individual Affymetrix GeneChip® Human Genome U133 Plus 2.0 arrays (total of 20 arrays) for 16 h at 45°C. Arrays were washed using an Affymetrix Fluidics Station and scanned using an Affymetrix GeneChip Scanner 3000 at a resolution of 3 μm. Normalization and computation of expression values were performed using the robust multichip average method (21). From the global gene expression matrix, which contains the expression values of the 54′675 probe sets at all time points, the expression values at baseline and after 1 h of sevoflurane inhalation (peak transcriptional change) were selected and used to determine: 1) differentially expressed genes using the Significance Analysis of Microarrays (SAM) algorithm (22) and 2) differentially expressed pathways using Gene Set Enrichment Analysis (GSEA) 23, as previously described in detail (20,24). In SAM, each gene is assigned a score on the basis of its change in gene expression relative to the standard deviation of repeated measurements for that gene. Genes with scores more than a threshold are deemed potentially significant and the algorithm provides an estimate of the false discovery rate (FDR), i.e., the expected proportion of false positives among the transcripts called significant. Conveniently, GSEA aggregates the per gene statistics across genes within a gene set, thus making it possible to detect situations where the genes in a predefined set change in a small but coordinated way. GSEA calculates an enrichment score (NES) for a given gene set using a ranked list of all genes and infers statistical significance (expressed as a P value and a FDR) of each NES against NES background distribution calculated by permutation of the original data set. A cut-off median FDR of 3% was used in SAM analysis, and P < 0.05 was used in GSEA analysis to obtain the ranked lists of differentially expressed genes and pathways, respectively. To define a standardized measure of gene expression over time (the mean centroid), we normalized the gene expression levels of the top up- and down-regulated genes to a mean of 0 and a variance of 1 across all 20 samples over time. Microarray analysis was performed for four individuals only to decrease related costs. All other analyses were performed for all five study subjects.

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Transcript Validation Using Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for four randomly selected genes to confirm microarray data (please see Supplementary Table S1 available at www.anesthesia-analgesia.org), as previously described (20,25). First strand cDNA was synthesized from 1 μg of total RNA using Superscript II reverse transcriptase (Invitrogen, Basel, Switzerland) and oligo-dT as primer. RT-PCR quantification of the selected genes was performed on a Stratagene MX3000 real-time sequence detector instrument (Stratagene Europe, Amsterdam, The Netherlands) using the Brilliant SYBR green QPCR Master Mix (Stratagene Europe). Amplification reactions were conducted with an initial step at 90°C for 3 min followed by 20–35 cycles. All PCR reactions were performed in triplicates, and ribosomal 18S (a constitutively expressed gene) was used as reference control. Predicted size of PCR products was confirmed by agarose gel electrophoresis.

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Determination of L-Selectin (CD62L) and β2-Integrin (CD11b) Expression in the Blood Using Flow Cytometry

L-selectin (CD62L) is an early indicator of neutrophil activation, while β2-integrin (CD11b) expression is a later event of the leukocyte-endothelial interaction. Collected heparinized blood samples were immediately processed for flow cytometry. The expression of the adhesion molecules was determined at baseline, 24, 48, and 72 h after sevoflurane exposure in all five volunteers. Two microliters of 0.5 mM N- formyl-methionyl-leucyl-phenylalanin (fMLP) was added to 1 mL of collected whole blood (1 μM final fMLP concentration) and incubated for 10 min at room temperature. Unstimulated blood served as control. Subsequently, 3 μL of the primary fluorochrome-labeled antibody was added to 50 μL of blood in endotoxin-free tubes and incubated in the dark for 10 min at room temperature. Lysis buffer (450 μL) (Becton Dickinson, Basel, Switzerland) was added and incubated for an additional 20 min at room temperature. The lysates were fixed for 30 min in 0.5 mL 0.2% paraformaldehyde solution at room temperature. The samples were centrifuged, and the cell pellets were suspended in 0.5 mL of TLR buffer, and stored at 4°C in the dark. The FACSCalibur (Becton Dickinson) flow cytometer was used to measure R-phycoerythrin (PE)-fluorescence at 580 nm and FITC-fluorescence at 515 nm. White blood cells were distinguished from each other by typical physical characteristics, resulting in well-delineated cellular subpopulations that are easily identified on forward and side-scatter plots. Monoclonal antibodies for polymorphnuclear granulocytes (CD15, PE-labeled, clone 80H5, Immunotech, Marseille, France) and monocytes (CD14, FITC-labeled, clone 61D3, eBioscience, Wembley, United Kingdom) further served for identification of cellular subgroups. CD62L and CD11b expression were measured by fluorescence intensity of PE-conjugated monoclonal antibodies directed against CD62L (FITC-labeled, clone DREG56, eBioscience) and CD11b (PE-labeled, clone 2LPM19C, DAKO, Glostrup, Denmark). Results were compared to isotype-matched control IgG (PE-labeled IgG, eBioscience and FITC-labeled IgG, Becton Dickinson). A minimum of 20,000 events was counted on each sample.

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Statistical Analysis

Data are given as mean (SD). Repeated-measures ANOVA were used for comparison. Student-Newman-Keuls test was used for post hoc analysis. P < 0.05 was considered significant. Analyses were performed using StatView Version 5 (SAS Institute, Chicago, IL).

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RESULTS

The 60 min period of sevoflurane inhalation at sedative concentrations (0.5%–1%) had only marginal effects on arterial blood pressure and end-tidal CO2 concentrations (Table 1). The participants were responsive to verbal commands or tactile stimuli at all times. The Bispectral Index ranged between 68 and 82 on average. All subjects tolerated the procedure without complications.

Table 1

Table 1

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Sevoflurane Inhalation at Subanesthetic Concentrations Markedly Altered Gene Expression in the Blood of Healthy Volunteers

We first tested whether sevoflurane at subanesthetic concentrations is capable of modulating gene expression in humans. For this purpose, mRNA from whole blood was isolated at different time points after sevoflurane application and used for gene chip hybridization. Gene chip data were independently confirmed with RT-PCR, which showed the same results (please see Supplementary Table S2 available at www.anesthesia-analgesia.org). Significance Analysis of Microarrays (SAM) revealed 74 upregulated (changes in expression more than 35% and median FDR <3%) and 63 down-regulated transcripts (median FDR <3%) after 60 min of sevoflurane exposure (for complete list of transcripts, please see Supplementary Table S3 available at www.anesthesia-analgesia.org). Three volunteers exhibited peak gene regulations after 1 h, while one subject exhibited peak transcriptional responses after 15 min of inhalation (Fig. 2). Using microarrays covering the entire human genome (>54,000 probes for the approximately 30,000 human genes), our data confirm the profound effects of volatile anesthetics on the transcriptome, as previously reported in rat hearts (24,25), and further extend these findings to human blood in response to subanesthetic concentrations of sevoflurane.

Figure 2

Figure 2

Figure 2

Figure 2

Our analysis detected a number of up- and down-regulated transcripts already known to be involved in the protection of the heart or other vital organs (Fig. 3; please see Supplementary Table S3 available at www.anesthesia-analgesia.org). In particular, these included the up-regulated transcripts paraoxonase, 12-lipoxygenase, DnaJ (Hsp40), and the down-regulated transcripts chemokine ligand 5 (a potent neutrophil chemoattractant, also called epithelial neutrophil activating protein-78) and phosphodiesterase 5A. Control experiments with inhalation of 50% oxygen for 1 h did not show any transcriptional regulation of paraoxonase, 12-lipoxygenase, and acyl-CoA synthase (Fig. 4). Sevoflurane inhalation down-regulated transcripts involved in peroxisome proliferator-activated receptor (PPAR) regulation and fatty acid oxidation in blood cells. We have recently shown that sevoflurane anesthesia down-regulates PPARα and its coactivator protein, PGC-1α, which transcriptionally regulate metabolism and lead to a shift in fuel preference away from fatty acid oxidation in the heart (20). In the present study, GSEA confirmed the coordinate down-regulation of genes involved in fatty acid oxidation after sevoflurane inhalation (NES = −0.55, P = 0.00, FDR q-value = 0.08) in blood cells, which closely correlated with PPAR-related genes (PGC-1α pathway: NES = −1.22, P = 0.02, FDR q-value = 0.43) (Figs. 5A–C). Finally, a large number of regulated transcripts emerged with yet unknown function in the context of organ protection (please see Supplementary Table S3 available at www.anesthesia-analgesia.org).

Figure 3

Figure 3

Figure 4

Figure 4

Figure 5

Figure 5

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Sevoflurane Inhalation Reduces L-Selectin (CD62L) Expression on Leukocytes and Induces Cellular Resistance to Inflammatory Stimulation 24–48 h After Exposure in Humans

To elucidate whether sevoflurane inhalation modulates protein expression consistent with a “second window” of preconditioning, we determined the expression of the important adherence molecules β2-integrin (CD11b) and L-selectin (CD62L) from cubital blood collected up to 72 h after sevoflurane application. Expression levels were determined separately on granulocytes (CD14 positive) and monocytes (CD15 positive). Sevoflurane administration significantly reduced (approximately 25%) the expression of L-selectin (CD62L) in granulocytes (P = 0.004), but had only a marginal effect on monocytes (Figs. 6A and B). These changes were most evident at 24 and 48 h after inhalation. In contrast, there was no decrease in β2-integrin (CD11b) expression in monocytes and granulocytes (data not shown). Importantly, granulocytes stimulated with the bacterial oligopeptide fMLP exhibited markedly reduced shedding of L-selectin (CD62L) (Fig. 6C and D, P = 0.009), indicating resistance to inflammatory stimulation. L-selectin (CD62L) shedding was maximally inhibited 24 to 48 h after sevoflurane exposure.

Figure 6

Figure 6

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DISCUSSION

The salient findings of the present study are as follows. First, sevoflurane inhalation, even at low subanesthetic concentrations, rapidly altered the blood transcriptome on a genome-wide scale in healthy subjects, a prerequisite for late preconditioning, which depends on de novo protein synthesis. The observed transcriptional changes specifically involved genes with known biological significance in the context of late preconditioning or organ protection. In accordance with our previous findings in human hearts exposed to sevoflurane (20), genes involved in fatty acid oxidation, regulated by the PCG1α-pathway, were similarly down-regulated in the blood. Second, 24 to 48 h after sevoflurane exposure, i.e., consistent with the occurrence of a late or second window of preconditioning, the expression of L-selectin (CD62L), a key inflammatory adhesion molecule responsible for the tethering of leukocytes to the endothelium (17), was reduced by approximately 25% on granulocytes, which further exhibited an increased resistance to inflammatory stimulation. Taken together, using blood as a model system and standardized experimental conditions, we provide for the first time molecular evidence that the anesthetic gas, sevoflurane, can induce late preconditioning in humans. Clearly, our findings should be confirmed in the clinical setting with patients.

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Animal Models of Late Preconditioning by Volatile Anesthetics

The potential of late preconditioning to provide enduring protection (lasting 30-fold longer compared to early preconditioning) is of potential clinical importance. However, it was elusive whether late preconditioning, i.e., protection developing 24 to 48 h after the application of the preconditioning stimulus, would also occur in humans in response to volatile anesthetics. Animal studies indicate that volatile anesthetics offer protection from ischemia-reperfusion injury in heart (12) and brain (26) tissue lasting for up to 48 h. Tonkovic-Capin et al. (13) first reported successful late preconditioning of the heart in a rabbit model using isoflurane at 1% end-tidal concentration. Subsequent studies confirmed these findings in rats (10–12) and mice (9). Interestingly, emulsified formulations of volatile anesthetics equivalent to only 0.2 MAC, and with no apparent sedation markedly reduced infarct size in rabbit hearts when administered IV 24 h before test ischemia (15). Signaling pathways involved in volatile anesthetic-induced late preconditioning are reminiscent of those observed in other types of late preconditioning (4,5), including reactive oxygen and nitrogen species as triggers, and 12-lipoxygenase, cycloxygenase-2, and inducible nitric oxide synthase (iNOS) as mediators or effectors, and the magnitude of the afforded protection is similar. In accordance with these animal studies, the present study now provides, for the first time, evidence of a late protective phenotype after sevoflurane inhalation in humans.

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Genetic Reprogramming by Sevoflurane: A Prerequisite for Late Preconditioning

Late preconditioning directly depends on transient transcriptional or translational changes (for h), and the delayed and prolonged protection is consistent with altered protein expression (for days). To test our hypothesis, we used the easily accessible blood compartment as a human model system, and determined the time course of transcriptional changes after sevoflurane inhalation in healthy volunteers. No transcriptional changes were measured after inhalation of 50% oxygen alone. However, genes encoding key protective proteins were significantly regulated after sevoflurane inhalation within only a short period, consistent with results obtained in humans after remote ischemic preconditioning of the limb (27). The observed changes in gene expression were only transient. Peak transcriptional changes occurred within 1 h of sevoflurane inhalation, but most of the changes disappeared after 24 h. This time course is characteristic for the transcriptional changes underlying the second window of protection in the heart. Salloum et al. reported peak iNOS mRNA expression in mice already 1–2 h after a single dose of sildenafil, a late preconditioning-inducing drug (4). Subsequently, the increase in iNOS mRNA completely vanished, but changes in cardioprotective iNOS protein expression were not measured before 24 h after the application of the triggering drug (4). This contention is consistent with our findings of an early transient transcriptional response (within hours) followed by late more persistent changes in protein expression (24–48 h later). Of note, changes in mRNA and protein expression were only modest (30%–40% compared to baseline) similar to our human study, but had marked effects on infarct size (from 30% to 6% of the area at risk). Interestingly, 12-lipoxygenase, which was recently implicated in delayed cardiac protection in mice (9), was upregulated. Conversely, phosphodiesterase 5A was down-regulated by sevoflurane. Inhibition of phosphodiesterase 5A by sildenafil was previously shown to induce late preconditioning in mice through the iNOS pathway (4). Additional potentially protective transcripts that were regulated in the blood transcriptome included the antioxidant paraoxonase (28), heat shock protein 40, known to prevent cytochrome c release from mitochondria, and a number of transcripts with less well-established function in the context of late preconditioning, such as EGR1, a master switch coordinating upregulation of stress genes, or NR1D2, a nuclear hormone receptor acting as a link between metabolism and inflammation (29). Collectively, our data show that sevoflurane inhalation reprograms blood transcriptome toward a defensive phenotype in humans.

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Sevoflurane-Induced Delayed Protection Against Leukocyte-Endothelial Activation

Leukocytes have a central role in the pathogenesis of ischemia-reperfusion damage. In the present study, we show that inhalation of sevoflurane decreases the expression of L-selectin (CD62L) in granulocytes and attenuates their responsiveness to inflammatory stimulation 24 to 48 h after exposure, consistent with a second window of protection. Extravasation of leukocytes is mediated by adhesion molecules, and exacerbates tissue injury after restoration of blood supply to the site of ischemia (30,31). In the dynamic interaction between leukocytes and endothelium, the cell surface receptor L-selectin (CD62L) and its proteolytic shedding mediate the initial steps of leukocyte tethering and rolling, while β2-integrin (CD11b) facilitates subsequent firm adhesion and transmigration (17). L-selectin (CD62L) is constitutively expressed on the surface of leukocytes. Inflammatory stimulation via G-protein coupled receptors, as mimicked in our study by the chemoattractant fMLP, rapidly activates L-selectin (CD62L) by phosphorylation and subsequent cleavage of its ectodomain, increasing the binding activity to the endothelium (17). Thus, our findings suggest that sevoflurane induces a resistance of leukocytes against noxious stimulation. Blockade of selectin-mediated leukocyte adhesion improves post-ischemic function in the heart (32), while genetically engineered mutations that prevent shedding of cell surface receptors markedly diminish the deleterious inflammatory response (33). In contrast to remote ischemic preconditioning of the limb, in our study small-dose sevoflurane did not decrease β2-integrin (CD11b) expression or its shedding in response to the chemoattractant fMLP. This is in agreement with previous in vitro experiments showing that 0.5 MAC isoflurane inhibited shedding of L-selectin (CD62L), but not β2-integrin (CD11b), in response to chemoattractants, whereas 1 MAC isoflurane affected both adhesion molecules (34). Although L-selectin (CD62L) was not among the differentially regulated transcripts in our human experiments, calmodulin, a calcium regulatory protein, which co-precipitates with L-selectin (CD62L) and tightly regulates its surface expression and function (35), was markedly down-regulated after sevoflurane inhalation (Fig. 5). This notion raises the interesting possibility that delayed alterations in L-selectin (CD62L) expression and function might be indirectly caused by reduced calmodulin expression. It has been traditionally thought that the biological effects of fast-acting anesthetic gases with low blood-gas solubility dissipate rapidly after exhalation. In contrast to this belief, our study clearly demonstrates that these gases continue to exert their potentially protective actions long after their physical clearance from the human body, consistent with published results in patients undergoing coronary artery bypass grafting surgery (36).

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Gene Regulatory Control of Energy Metabolism in the Blood Mirrors Conditions in the Heart

We have recently shown that IV and volatile anesthetics differentially regulate the transcriptional response to cardiac surgery in human hearts (20). In particular, sevoflurane shifted the metabolic fuel preference away from fatty acid oxidation, which closely correlated with improved postoperative cardiac function. Myocardial substrate metabolism critically affects cardiac function (37), and switching metabolic fuel preference away from fatty acid oxidation improves recovery after ischemia and even affects long-term outcome (38). The present study confirms similar metabolic changes in the blood after subanesthetic sevoflurane inhalation, and further suggests that the transcriptional changes in fatty acid oxidation may be due to down-regulation of the PGC-1α pathway. This nuclear receptor coactivator critically controls cellular energy metabolism, and thus determines oxygen consumption (39). Moreover, inhibition of the PGC-1α pathway was recently shown to reduce uptake of oxidized low-density lipoprotein into macrophages and to prevent their retention in atherosclerotic vessel walls, thereby stabilizing vulnerable plaques (40). Hence, modulation of the PGC-1α pathway, as observed after sevoflurane administration, may be a novel antiischemic and plaque-stabilizing strategy in perioperative medicine.

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Limitations

The sample size of this study is small. However, a highly standardized protocol was used in healthy volunteers to overcome confounding variables. In this study, we used ranitidine to protect the participants from the potential hazard of pulmonary aspiration, and some previous reports suggest a role of histamine receptors in modifying expression of adhesion molecules (41,42). However, there was no transcriptional regulation in our control experiments after inhalation of 50% oxygen and administration of ranitidine alone. Also, changes in L-selectin expression exhibited the maximum at 24–48 h after sevoflurane inhalation. At this time, a single dose of ranitidine is cleared from the body, making a relevant effect on adhesion molecules unlikely. Moreover, the H2 receptor antagonist, ranitidine, did not affect histamine-induced expression of adherence molecules (42), nor did it prevent histamine-induced leukocyte rolling (41). Future studies should help to precisely delineate the underlying signaling components. Whether the detected molecular evidence of late preconditioning by sevoflurane successfully translates into clinical benefits requires additional randomized controlled trials.

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CONCLUSIONS

This study provides molecular evidence of late preconditioning after sevoflurane inhalation in healthy volunteers. Because the late phase lasts significantly longer than the early phase, its protective potential merits further investigation in the clinical arena.

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ACKNOWLEDGMENTS

The authors thank the PACU nurses, colleagues, and volunteers, who participated in this study.

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