Characterization of the Postconditioning Effect of Dexmedetomidine in Mouse Organotypic Hippocampal Slice Cultures Exposed to Oxygen and Glucose Deprivation

• ❖ General anesthetics reduce ischemic brain injury in animals when administered after the ischemic event, but whether the common sedative dexmedetomidine does so is not known

• ❖ In brain slices from mice, dexmedetomidine reduced injury from oxygen and glucose deprivation when administered after the deprivation, thereby showing postconditioning protection

• ❖ Dexmedetomidine may be neuroprotective even when administered after cerebral ischemia

PERIOPERATIVE acute ischemic stroke remains an important factor source of morbidity and mortality.1We have previously shown that dexmedetomidine, a potent and selective agonist of the α2-adrenoceptors with anesthetic, analgesic, and brain-protective properties,2–5exerts an early preconditioning effect in the acute rat hippocampal slice subjected to oxygen and glucose deprivation (OGD).6This means that preexposure of brain tissue to dexmedetomidine resulted in a less severe tissue injury after OGD application.7–10This effect was mediated in part via  activation of the nonreceptor tyrosine kinase focal adhesion kinase (FAK) stimulated by the α2-adrenoceptors. When phosphorylated, FAK activates the survival protein Akt by a cellular cascade involving the Src kinases and the phosphatidyl-inositol 3 kinase (PI3-kinase).11,12Dexmedetomidine also increases hippocampal phosphorylated extracellular signal-regulated protein kinase 1 and 2 (ERK1&2) protein expression, a key enzyme involved in coupling cellular signaling to long-term phenomena such as neuroprotection.13This effect is mediated by mechanisms independent from the α2-adrenoceptor signaling cascade and most likely an imidazoline 1 receptor-protein kinase C pathway. Among the downstream survival signals elicited by ERK 1&2, the ATP-dependant mitochondrial K channels (MitoK^ATP) have been shown to play a role in cell survival in both neuronal and extraneuronal tissues.14,15 

Several lines of evidence suggest that reduction of ischemic brain injury can also be obtained by postconditioning. Pharmacologic postconditioning consists of applying the neuroprotectant after the occurrence of the ischemic event, which results in a decrease in the severity of tissue injury elicited by a more prolonged ischemic challenge. Postconditioning may be particularly clinically relevant, because interventions can be delivered after the onset of brain ischemia. Interestingly, postconditioning has been recently reported for volatile anesthetics because isoflurane was shown to protect the brain tissue against ischemic insult when applied after the ischemic stimulus.16 

The aim of this study was to examine whether dexmedetomidine exhibits postconditioning effects against injury induced by OGD in mouse hippocampal organotypic slice cultures. Further, the role of α2-adrenoceptor-FAK-PI3-kinase-Akt and imidazoline I1-receptor-ERK1&2-MitoK^ATPpathways in these effects was studied.

Handling procedures according to the Guide for the Care and Use of Laboratory Animals were followed throughout. Experiments were performed on 3-day-old OF1 mice (Iffa-Credo, L'Arbresle, France). Approval was obtained from the Institutional Animal Care and Use Committee at Paris VII University (Paris, France).

This study, including care of the animals involved, was conducted according to the official edict presented by the French Ministry of Agriculture (Paris, France) and the recommendations of the Helsinki Declaration. Thus, these experiments were conducted in an authorized laboratory and under the supervision of an authorized researcher (Pierre Gressens, MD, PhD, INSERM U 676. Paris, France).

The following agents were studied alone or in combination: Dexmedetomidine (1 μm), PD 098059 (5 μm, an inhibitor of mitogen-activated kinase [MEK] 1&2, the direct activator of ERK1&2; Sigma, St-Quentin Fallavier, France), yohimbine (100 μm; Sigma), the inhibitor of Src tyrosine kinase 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2, 5 μm; Calbiochem, Nottingham, United Kingdom), wortmannin (100 nm,17an inhibitor of phosphatidyl inositol 3 Kinase-Akt, PI3K-Akt pathway; Sigma), efaroxan (an inhibitor of the imidazoline1 receptors, 10 μm; Sigma), chelerythrine (an inhibitor of proteins kinases C, 10–5 m; Sigma), the analog of cyclic adenosine monophosphate (8 bromo cycline monophosphate, 4 × 10⁻³m; Sigma), the inhibitor of phosphor-diesterases 3-isobutyl-1-methylxanthine (10×⁴m; Sigma), 5-hydroxydecanoic acid (5HD, 10 μm, an inhibitor of the mitochondrial ATP dependent K channels; Sigma), and antiproteases (50 μg/ml leupeptin, 10 μg/ml aprotinin, and 5 μg/ml pepstatin; Sigma).

Hippocampal organotypic slice cultures were prepared according to the method described by Stoppini et al.  modified by others.18–20Hippocampi were dissected from the brains of 5-day-old OF1 mice pups (Iffa Credo, L'Arbresle, France). Brains, aseptically removed, were placed in an ice-cold solution containing minimum essential medium without glutamine and NaHCO3, supplemented with 3 mm l-glutamine, 19 mm glucose, 30 mm Hepes, 5 mm NaHCO3, 0.5 mm l-ascorbic acid, 0.7 mm CaCl2(H2O)2, 1.4 mm MgSO4, heat-inactivated horse serum 25% (Sigma), and pH 7.3. Coronal sections (400 μm) were cut from each hemisphere using a McIIwain tissue chopper. Hippocampal slices were rapidly dissected from whole slices in an ice-cold preparation medium. Slices were than transferred to 30-mm Millicell-CM tissue culture inserts (Millipore Corp., Sigma), which were prebuffered with medium in 6-well plates in a moist 5% CO²atmosphere at 37°C for 60 min. Three hippocampal slices were placed on each insert and maintained in vitro  for 10 to 14 days. Medium was changed three times a week.

Ischemia was simulated in vitro  by OGD. Slices were transferred into an hermetic glass beaker containing glucose-free artificial cerebrospinal fluid flowed with 5% CO²and 95% N²and immersed in a water bath servocontrolled to 37°C. Partial oxygen pressure was monitored during deoxygenation of the beaker. Partial oxygen pressure was accessed using the S/5 Compact Anesthesia Monitor (GE Healthcare, Wauwatosa, WI). When partial oxygen pressure reached 0 (an average 10-min period of time was necessary to achieve this stage), all the air entries were closed, and the beaker containing the slices was placed in the incubator chamber at 37°C for the desired period of OGD for the desired period of time of OGD (10, 20, 30, or 60 min). At the end of this period, partial oxygen pressure was checked to ensure that it was still equal to 0 and pH, and partial oxygen and carbon dioxide pressures were measured in the media. Slices were then recovered during 72 h in physiologic conditions.

In the postconditioning experiments, the optimal duration of the reperfusion time (time between the end of ODG and the administration of dexmedetomidine associated with the maximum protective effects of dexmedetomidine) was selected, and the postconditioning experiments were conducted with various concentrations of dexmedetomidine (10⁻⁸to 10⁻⁴m, 30 min). Slices subjected to OGD were treated (or not) with dexmedetomidine (1 μm, 30 min) in a physiologic-oxygenated medium by using various times after reperfusion (10, 30, 60, 90, and 120 min). The 1-μm dexmedetomidine concentration was used with or without various pharmacologic modulators of α2-adrenoceptors-FAK-Src-PI3-kinase-Akt and the imidazoline1 receptors-ERK 1&2-mitochondrial ATP-dependent K channel pathways (given 30 min before and during dexmedetomidine challenge). Preconditioning experiments with dexmedetomidine were also conducted to examine whether preconditioning and postconditioning may share common mechanisms. In the preconditioning experiments, slices were first treated with various dexmedetomidine concentrations alone (10⁻⁸to 10⁻⁴m, 30 min). After 3 h of free interval in normal culture media, slices were submitted to OGD. Slices were than submitted to the dexmedetomidine concentration corresponding to the EC⁵⁰value alone or in combination with pharmacologic modulators of FAK and ERK1&2 signaling cascades given 30 min before and during dexmedetomidine challenge.

Slices were submitted to a 5-min dexmedetomidine challenge (EC⁵⁰value for dexmedetomidine-induced preconditioning effect) alone or in combination with pharmacologic modulators of the α2-adrenoceptor-FAK-Src-PI3-kinase-AKt and of the imidazoline I1-ERK1&2-MitoK^ATPpathways. Agents supposed to increase FAK and ERK1&2 phosphorylation were applied for 5 min. Agents supposed to block dexmedetomidine (or any other activator)-induced changes in phosphorylated FAK and ERK1&2 expression were administered 30 min before dexmedetomidine (or any other activator). At the end of the experiments, cerebrospinal fluid was aspirated, slices were frozen in liquid nitrogen, and then homogenized by sonication in 200 μl of a solution of 1% (wt/vol) sodium dodecyl sulfate, 1 mm sodium orthovanadate, and antiproteases (50 μg/ml leupeptin, 10 μg/ml aprotinin, and 5 μg/ml pepstatin) in water at 100°C and placed in a boiling bath for 5 min. Homogenates were stored at −80°C until processing.

Protein concentration in the homogenates was determined with a bicinchoninic acid-based method, by using bovine serum albumin as the standard. Equal amounts of protein (30 μg) were subjected to 6% (wt/vol, ERK 1 and 2) or 13% (wt/vol, FAK) polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and transferred electrophoretically to nitrocellulose. For detection of active phosphorylated forms of ERK1&2, immunoblot analysis was performed with affinity-purified rabbit antidiphosphotyrosine antibodies (threonine 202/tyrosine 204 monoclonal mouse IgG, clone 4G10; Euromedex 05-321, Souffelweyersheim, France), and total ERK immunoreactivity was detected using total ERK antibodies (cell signaling, diluted at 1/1000). Detection of phosphorylated active forms of FAK was performed using rabbit anti-Y397 FAK phosphospecific antibody (Biosource International, Camarillo, CA; diluted at 1:1000). Identification of total FAK was made with an anti-FAK antibody directed against the nonphosphorylated residues of the protein (Biosource International; diluted 1:1000). Primary antibodies were labeled with peroxidase-coupled antibodies against rabbit IgG, which were detected by exposure of molecular probe autoradiographic films in the presence of a chemiluminescent reagent (ECL, Amersham, Little Chalfont, United Kingdom). The specificity of the immunoreactivity for ERK 1&2 was assessed by its competition in the presence of 50 μm o-phosphotyrosine. Variations between gels were controlled by expressing the results as a percentage of increase or decrease from control of the ration of phosphorylated proteins on total forms of the same protein (Cohu High Performance CCD camera, Gel Analyst 3.01 pci, Paris, France). For each band, blank values were subtracted before calculating the ratio.

Quantification of cell death was performed using two methods: detection of active cleaved caspase 3 by Western blot analysis and propidium iodide (PI) fluorescence.21,22For detection of active caspase 3, immunoblot analysis was performed with the rabbit polyclonal IgG anticaspase 3-specific antibodies detecting both the 32 Kd entire protein and the 17-Kd fragment produced by cleavage of caspase 3 when activated, as previously used in our laboratory6(Upstate biotechnology diluted 1:2000, Euromedex). The 17-Kd band immunoreactivity was considered cleaved caspase 3 and taken into consideration in the statistical analysis. Immunoreactive bands normalized to β-actin band were quantified using specific monoclonal antiactin A5316 antibody (Sigma) by using a computer-assisted densitometer and expressed as a percentage of increase from control. For each band, blank values were subtracted before calculating the ratios.

Quantification of cell death was detected using fluorescent PI in the CA1 and dental Gyrus subfield areas of the hippocampus (Invitrogen, Cergy Pontoise, France, P-3566). PI was added to the media from the beginning of the OGD to the end of the experiment until PI binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per four to five pairs of DNA bases. Once the dye is bound to the nucleic acids, its fluorescence is enhanced 20- to 30-fold, the maximal absorption wave for PI being 535 nm and the maximal fluorescence emission wave being 617 nm. PI was added to the media (3 μm) from the beginning of OGD to the end of the experiment in three independent experiments. Stained cells were examined using a fluorescence microscope equipped with an appropriate filter (UV-2A; Zeiss, Oberkochen, Germany; excitation, 530 nm; emission, > 600 nm) and images were digitalized. All the slices were analyzed the same time by an observer unaware of the treatment assignment. For each slice, PI fluorescence of 10 areas from each subfield area of the hippocampus were analyzed using Image J 1.31v software (National Institutes of Health, Rockville Pike Bethesda, MD).

For Western blot analysis, data were collected from 10 independent experiments run in triplicate (each independent experiment was reproduced 10 times). Each independent experiment was performed with one animal (n = 30). For PI fluorescence, three independent experiments were used for each condition. In each experiment, three slices were used, and fluorescence from 10 random areas were analyzed in these three slices (n = 90 for each condition). After the normality of data were accessed by Shapiro-Wilk W test, statistical analysis was performed by one-way ANOVA followed by post hoc  analysis using Student t  test with Bonferroni correction (according to the number of post hoc  comparison necessary) generated using the GraphPAD 4.0 software (Intuitive Software for Science, San Diego, CA). Results are expressed as mean ± SD. P < 0.05 was considered the threshold for significance. Concentration-response curves and statistical analysis were generated using the GraphPAD 4.0 software. The functions used to fit the curves to the data were the following four-parameter logistic equation: Y =A + (B −A )/(1 + 10^{(LogEC50−X)}), where A  is the minimum, B  is the maximum, EC⁵⁰is the fitted 50% effect concentration, and X  is the logarithm of the concentration.

During OGD challenge, Po²measured in the medium was always less than 5 mmHg (3 ± 1 mmHg), whereas Pco²was 39 ± 2 mmHg and pH was 7.4 ± 0.01. Cell death measured by PI fluorescence in CA1 and dental gyrus, 72 h after OGD increased with the duration (10, 20, 30, and 60 min) of OGD with a ceiling occurring between 60 and 90 min. Therefore, 60 min was chosen as the duration of the OGD period applied to slices in all experiments (fig. 1).

To determine the optimal postconditioning timing, dexmedetomidine (10⁻⁶M) was applied 10, 30, 60, 90, and 120 min after the end of the 60 min OGD. Under these conditions, the neuroprotective effect of dexmedetomidine was maximum for the 60 min reperfusion period after the end of OGD, with a ceiling effect between 60 and 120 min (fig. 2). Consequently, the 60-min delay was selected before administering dexmedetomidine (1 μm) as a postconditioning stimulus.

Postconditioning induced by dexmedetomidine was limited in magnitude and could not be fitted by the model to provide an EC⁵⁰value. However, a ceiling effect was observed for dexmedetomidine concentrations between 10⁻⁶and 10⁻⁴m (fig. 2). The postconditioning effect of dexmedetomidine (10⁻⁶m) was found present only in the CA1 subfield area, where dexmedetomidine postconditioning significantly decreased PI-labeled cell death (fig. 3). These effects were markedly reduced either by the application of yohimbine (100 μm), PP2 (5 μm), or wortmannin (100 nm) in the CA1 hippocampal subfield area (fig. 3) or by efaroxan (10 μm), PD 08059 (5 μm), or 5HD (10 μm) in both CA1 and dental gyrus hippocampal subfield areas (fig. 3). In addition, the protein expression of cleaved caspase 3 was significantly decreased by dexmedetomidine postconditioning in comparison with control conditions (fig. 4). The effect of the combination of either PP2 plus wortmannin or PD 098059 plus 5HD was not significantly different from the effect of a single of the two agents in each combination. In contrast, the combination of either PP2 plus PD 098059 or wortmannin plus 5HD completely abolished the postconditioning effect of dexmedetomidine measured by either PI fluorescence or expression of cleaved caspase 3. Taken alone, yohimbine, PP2, wortmannin, efaroxan, PD 08059, or 5HD have no protective effect on cell death during physiologic condition or against OGD-induced cell death labeled by PI or active caspase 3 expression (data not shown).

To simplify the presentation of the results, only the key findings on dexmedetomidine-induced preconditioning are reported here. Unlike postconditioning, preconditioning was observed in both the CA1 and dental gyrus hippocampal subfield areas. No significant difference in dexmedetomidine effects was noted between these two areas; therefore, the data presented originate all from the CA1 subfield. Dexmedetomidine induced a concentration-related preconditioning effect (EC⁵⁰value = 7 × 10⁻⁷M, 95% confidence interval 2 × 10⁻⁷to 2.5 × 10⁻⁶m; fig. 5). Dexmedetomidine (1 μm) administered 3 h before 60 min OGD significantly decreased both PI-labeled cell death and protein expression of cleaved caspase 3 (figs. 6 and 7). As was the case for postconditioning, the preconditioning effect of dexmedetomidine on both PI-labeled cell death and cleaved caspase 3 expression was markedly reduced by the application of yohimbine (100 μm), PP2 (5 μm), or wortmannin (100 nm) and was also decreased by efaroxan (10 μm), PD 08059 (5 μm), or 5HD (10 μm). Combinations of pharmacologic agents similar to those used for postconditioning experiments gave similar results during preconditioning challenges (figs. 6 and 7). Taken alone, yahimbine, PP2, wortmannin, efaroxan, PD 08059, or 5HD have no protective effect on cell death during physiologic condition or against OGD-induced cell death labeled by PI or active caspase 3 expression (data not shown).

In physiologic conditions (without OGD challenge), the 1-μm concentration for dexmedetomidine was selected from the concentration used during neuroprotection challenges. Dexmedetomidine (1 μm) increased FAK and ERK1&2 protein expression in the hippocampal slices (153 ± 15.6%, P < 0.001 and 178 ± 17%, P < 0.001, respectively). The increase in FAK phosphorylation was sensitive to yohimbine 100 μm (105 ± 22%, P > 0.05 vs.  control) or adenylate cyclase stimulation by the association of 8 bromo cycline monophosphate P and 3-isobutyl-1-methylxanthine (96 ± 18%, P > 0.05 vs.  control), whereas that in ERK 1&2 expression was totally blocked by efaroxan (103 ± 9%, P > 0.05 vs.  control), chelerythrin (98 ± 6%, P > 0.05 vs.  control), or PD 098059 (102 ± 11%, P > 0.05 vs.  control).

The original findings of the current study are the following: we have shown that dexmedetomidine (1 μm) exerts postconditioning effects against OGD-induced injury in mouse organotypic hippocampal slice cultures. The α2-adrenoceptor-FAK-PI3-kinase-Akt and the imidazoline I1 receptor-ERK1&2-MitoK^ATPpathways are very likely to contribute to these effects, which indicate that postconditioning and preconditioning induced by dexmedetomidine share common mechanisms.

The postconditioning effect of dexmedetomidine reported in the current study represents the main original finding. The limited efficacy of postconditioning in terms of preventing cell death may explain in part that it was restricted to the most vulnerable area of hippocampus (CA1). Interestingly, dexmedetomidine's maximal postconditioning effect required a 60-min reperfusion period after cessation of OGD. This finding might be the consequence of cellular energy failure induced by OGD, which alters protein phosphorylation, an energy-dependent process. These findings are consistent with previous results showing that recovery field potential depression induced by OGD was delayed by 1 h in rat hippocampal slices.23In addition, Zalewska et al.  24found that a brief period of OGD applied to hippocampal slices to induced FAK (and Src kinases) dephosphorylation up to 30 min after the OGD challenge. Finally, we have previously shown that anesthetics protect FAK from OGD-induced dephosphorylation in an acute model of hippocampal slices.25In contrast, isoflurane postconditioning was found efficient immediately after the beginning of the reperfusion in rat corticostriatal slices subjected to 15 min OGD.16Several hypotheses may explain these differences: energy deprivation elicited by OGD was most likely to be lower for a 15-min OGD period than for a 60-min one. Alternatively, the cellular mechanisms leading to the activation of the postconditioning effectors might be different between isoflurane and dexmedetomidine.

Cell death measured by PI fluorescence and caspase 3 expression was significantly reduced by the α2-adrenoceptor antagonist yohimbine, the FAK-Src inhibitor PP2, and the PI3-kinase AKt inhibitor wortmannin. PI cannot discriminate between necrotic or apoptotic cell death, and we did not use direct markers of necrotic cell death such as lactate deshydrogenase release. However, possible antiapoptotic properties have been established for dexmedetomidine in ischemic brain rats.6,26 

To date, little is known about postconditioning mechanisms in the brain. We and others have shown that in addition to its actions on the α2-adrenoceptors, dexmedetomidine increases phosphorylation of ERK1&2.13,27–29A large body of recent work indicates that both ERK 1&2 and Akt pathways, named the survival kinases, contribute to postconditioning of the heart against ischemic injury.30–32However, activation of ERK1&2 had never been shown to mediate preconditioning or postconditioning actions of dexmedetomidine before. Recent data obtained on the brain tissue found that ERK 1&2, MitoK^ATP, and Akt were involved in brain postconditioning.16,33Our results show for the first time that both the α2-adrenoceptor-FAK-Src-PI3-kinase-AKt and the imidazoline I1 receptor-ERK1&2-MitoK^ATPpathways are involved in the postconditioning effects of dexmedetomidine against OGD-induced injury. Dexmedetomidine postconditioning was still observed in the presence of pretty efficient doses of antagonists/inhibitors of the α2-adrenoceptor-FAK-Src-PI3-kinase-AKt pathway. This supports that increasing concentrations of these agents could not further reduce dexmedetomidine postconditioning effect. Interestingly, cell death was also significantly reduced by the imidazoline I1 receptor antagonist efaroxan, the MEK1&2 inhibitor PD 098059, and the MitoK^ATPinhibitor 5HD used at high concentrations as well. These findings are consistent with our previous data showing that ERK1&2 phosphorylation is increased by dexmedetomidine (1 μm) via  a non-α2-adrenoceptor-dependent, protein kinase C-mediated, mechanism.13Postconditioning produced by dexmedetomidine was abolished by coadministration of either PP2 plus PD 098059 or wortmannin plus 5HD, which suggests that dexmedetomidine preconditioning effect proceeds via  activation of both α2-adrenoceptor-dependent and -independent mechanisms.

FAK and its downstream postconditioning effectors of the PI3K-Akt pathway have been shown to decrease apoptotic cell death by inhibition of Bad proapoptotic factor through AKt stimulation.34FAK activation has also been convincingly shown to promote cell survival in extraneuronal cells by inhibiting apoptosis.8,34ERK 1&2 is a key enzyme involved in coupling cellular signaling to long-term phenomena such as neuroprotection and cell survival.15,35Activation of the MitoK^ATPchannels depends on protein kinase C and ERK1&2 phosphorylation and is involved in the preconditioning effects of volatile anesthetics on cortical neurons in vitro .15,35Our results extend these findings by suggesting the involvement of the MitoK^ATPin dexmedetomidine-induced postconditioning in the mouse hippocampus. Whether dexmedetomidine directly activates these channels has not been examined yet.

Interestingly, our results indicate that the α2-adrenoceptor-FAK-PI3-kinase-Akt and the imidazoline I1 receptor-ERK1&2-MitoK^ATPpathways are involved in the preconditioning effects of dexmedetomidine in this model. Although we did not directly compare preconditioning and postconditioning potency in the current study, it can be speculated that the preconditioning effect of dexmedetomidine is more robust, at least owing to the lower EC⁵⁰value for dexmedetomidine and the larger hippocampal area protected by the preconditioning experiments. Our data indicate that both preconditioning and postconditioning induced by dexmedetomidine in this model might share common mechanisms (fig. 8). However, our results could not confirm these pathways because of the effects of the pharmacologic inhibitors used on other targets.

The use of organotypic slice cultures allowed us to demonstrate that dexmedetomidine brain-protective effects were still present 72 h after cessation of exposure to dexmedetomidine, which extends our previous findings in rats.6Cell death has been shown to peak at that time following OGD hippocampal injury in vitro .22,36This delay also allowed preserving the quality and viability of the slices, as shown in the control experiments with PI. We cannot speculate whether dexmedetomidine preconditioning or postconditioning effects are still perceptible after a longer period of time post-OGD. Here, we used the mouse instead of the rat for preparation of the hippocampal slices, because this approach leaves open the possibility of exploring the behavior of slices obtained from animals with selective targeted gene deletions or mutations. OGD-induced cell death was particularly important in the CA1 subfield of the hippocampus, whereas it was less pronounced in the dentate gyrus, which is consistent with previous findings.21,22,36Although we did not perform extensive measurement of apoptotic cell death, the increase in the expression of the 17-kd fragment of caspase 3, a key enzyme in the apoptotic cascade, suggests that caspase 3 was cleaved and activated in this model of ischemia. Brain caspase 3 expression has been found increased in neurons subjected to hypoperfusion.37Therefore, apoptotic cell death was likely to be present in our slice model.

We used a pharmacologic approach to investigate the cellular mechanisms involved in dexmedetomidine preconditioning and postconditioning effects. Dexmedetomidine-induced preconditioning effect was clearly concentration dependent. The EC⁵⁰value reported here is fairly consistent with those reported in our previous studies performed in rats.6,13Interestingly, there was a ceiling effect observed for this concentration of dexmedetomidine in postconditioning experiments. The 1-μm concentration tends to be greater than the mean plasma concentration reported after dexmedetomidine administration in humans (4–10 nm). However, plasma concentrations up to 1 μm can be reached after an intravenous administration.38,39This supports the physiologic relevance of our findings.

In conclusion, we have shown that dexmedetomidine exhibits postconditioning properties against hippocampal OGD-induced injury, which are still observed 72 h after OGD. The α2-adrenoceptor-FAK-PI3-Akt and the I1 receptor-ERK1&2-MitoK^ATPpathways are very likely to be involved in these effects, and in dexmedetomidine-induced preconditioning as well. These results could provide a working hypothesis to better understand some neuroprotective properties of this agent.

The authors thank Outi Mäki-Ikkola, Ph.D., Senior Researcher, Orionpharma, Turku, Finland, for the generous gifts of dexmedetomidine.