Characterization of the postconditioning effect of dexmedetomidine in mouse organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation.

BACKGROUND: There is an increasing interest in the use of dexmedetomidine for anesthesia and sedation. Here, we used the mouse organotypic hippocampal slice culture to investigate whether dexmedetomidine exhibits postconditioning properties against oxygen and glucose deprivation (OGD). The role of the focal adhesion and extracellular-regulated kinases pathways in these effects were examined in both postconditioning and preconditioning. MATERIALS AND METHODS: Slices were obtained from P5 mouse. In postconditioning experiments, Dexmedetomidine (1 microm) was incubated 60 min after the end of OGD. In preconditioning experiments, dexmedetomidine was applied 3 h before OGD. Pharmacologic modulation of the studied pathways was achieved by using selective inhibitors of these cascades. Cell death was assessed 72 h after OGD using propidium iodide labeling and protein expression of activated caspase 3. RESULTS: Maximum cell death increased with the duration of OGD. Dexmedetomidine induced a postconditioning effect in the CA1 (but not dentate gyrus) subfield area, which was significantly reduced by modulators of the focal adhesion and the extracellular-regulated kinases pathways. The combination of the inhibitors of the two pathways completely abolished the postconditioning effect of dexmedetomidine. The preconditioning effect of dexmedetomidine against ischemia-induced injury was observed in all hippocampal subfield areas. Results obtained with the pharmacologic modulation used for postconditioning also applied to dexmedetomidine-induced preconditioning. DISCUSSION: Dexmedetomidine exhibits significant, but moderate, postconditioning properties against oxygen and glucose deprivation-induced injury. Activation of focal adhesion and the imidazoline 1 receptors-extracellular-regulated kinases pathways is involved in dexmedetomidine-induced postconditioning and preconditioning as well.

The preconditioning effect of sevoflurane on the oxygen glucose-deprived hippocampal slice: the role of tyrosine kinases and duration of ischemia.

BACKGROUND: The neuroprotective efficacy of anesthetics observed in experimental models remains unproven in the clinical setting. The nonreceptor tyrosine kinase focal adhesion kinase (FAK) has been suggested to be involved in the neuroprotective effect of anesthetics observed experimentally. In the present work, we investigated whether FAK and the duration of ischemia play a role in the preconditioning effect of sevoflurane on brain tissue. METHODS: Rat acute hippocampal slices were subjected to oxygen and glucose deprivation (OGD) challenge during increasing periods of time (10, 20, 30, 45, 50, and 60 min) followed by 1 h reperfusion. A preconditioning sevoflurane concentration (10(-4) M, 1 h) was applied 3 h before initiation of OGD. Protein expression of FAK and cleaved caspase 3 (a marker of activation of the apoptotic cascade) was measured by immunoblotting. Cell death was assessed by propidium iodide (PI) fluorescence. RESULTS: Both PI fluorescence and expression of cleaved caspase 3 significantly increased with duration of ischemia until reaching a ceiling effect for durations of ischemia longer than 30 min. Sevoflurane (10(-4) M) increased FAK expression and markedly reduced the increase in PI fluorescence and cleaved caspase 3 expression for periods of ischemia of 10, 20, and 30 min. In contrast, the protective effect was no longer observed for periods of ischemia longer than 30 min. 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2, 10(-5) M, an inhibitor of src tyrosine kinases) application 60 min before and throughout that of sevoflurane significantly reduced the neuroprotective effect of sevoflurane on both caspase 3 expression and PI fluorescence. CONCLUSION: In the OGD rat acute hippocampal slice, the preconditioning effect of a clinically relevant concentration of sevoflurane was very likely to involve FAK and was observed only for periods of ischemia

Dexmedetomidine increases hippocampal phosphorylated extracellular signal-regulated protein kinase 1 and 2 content by an alpha 2-adrenoceptor-independent mechanism: evidence for the involvement of imidazoline I1 receptors.

BACKGROUND: Dexmedetomidine is a potent and selective alpha2-adrenoceptor (alpha2AR) agonist that exhibits a broad pattern of actions, including sedation, analgesia, and neuroprotection. Recent studies have emphasized the role of phosphorylated extracellular signal-regulated protein kinases (pERK1 and 2) in coupling rapid events such as neurotransmitter release and receptor stimulation long-lasting changes in synaptic plasticity and cell survival. Here, the authors hypothesized that dexmedetomidine increases pERK1 and 2 content and examined the mechanisms involved in this effect. METHODS: The effects of dexmedetomidine and their sensitivity to various pharmacologic agents on expression of pERK1 and 2 were studied by Western blots in hippocampal slices obtained from rats, wild-type mice, and mice carrying targeted deletions of the alpha2AR subtypes. RESULTS: Dexmedetomidine induced a concentration-related increase in the expression of pERK1 and 2 in rat hippocampal slices (EC50 [95% confidence interval] for pERK1, 0.97 [0.68-1.37] microm; for pERK2, 1.15 [0.62-2.14] microm). This effect was insensitive to the inhibitors of the alpha2AR-mediated signaling pathway, to prazosin, and to PP2, an inhibitor of the focal adhesion kinase-Src kinases. In contrast, it was still present in mice deleted for each of the alpha2AR subtypes and was markedly decreased by the antagonist of the I1-imidazoline receptors efaroxan, by phospholipase C and protein kinase C inhibitors, and by PD 098059, a direct inhibitor of ERK1 and 2 phosphorylation. CONCLUSION: Dexmedetomidine increases the expression of pERK1 and 2 via mechanisms independent of alpha2AR activation. The I1-imidazoline receptors likely contribute to these effects. The results may be relevant to some long-term effects (e.g., neuroprotective) of dexmedetomidine in the brain.

The effects of lidocaine and bupivacaine on protein expression of cleaved caspase 3 and tyrosine phosphorylation in the rat hippocampal slice.

Severe neurologic sequelae have been reported with the use of lidocaine after spinal anesthesia. This is considered a consequence of the high concentrations reached in the cerebrospinal fluid. We have previously shown that lidocaine increases the phosphorylation of focal adhesion kinase (FAK, a nonreceptor tyrosine kinase playing a role in neuronal plasticity and cell death). Here, we compared the effects of lidocaine and bupivacaine on FAK phosphorylation and cleaved caspase 3 expression in rat hippocampal slices. Slices were treated with increasing concentrations of lidocaine (4.3 nM to 4.3 mM) or bupivacaine (3.4 nM to 3.4 mM) in the presence or absence of the specific inhibitor of the FAK tyrosine kinase PP2 (10 microM). Caspase 3 expression and FAK phosphorylation were examined by immunoblotting. Lidocaine induced a concentration-related increase in FAK phosphorylation while the bupivacaine effect was biphasic. The maximal effect observed with millimolar lidocaine concentrations was significantly more than with clinically equipotent bupivacaine concentrations (4.3 x 10(-3) M lidocaine: 168% +/- 20%, mean value +/- sd; 10(-3) M bupivacaine: 145% +/- 19% P < 0.001). The expression of cleaved caspase 3 was increased by lidocaine, but not bupivacaine, at millimolar concentrations and was blocked by PP2. Our results indicate that millimolar concentrations of lidocaine, but not bupivacaine, increase cleaved caspase 3 expression. The role of FAK phosphorylation in this effect remains to be clarified.

Effects of dexmedetomidine on hippocampal focal adhesion kinase tyrosine phosphorylation in physiologic and ischemic conditions.

BACKGROUND: Dexmedetomidine is a potent and selective alpha2-adrenoceptor agonist that exhibits a broad pattern of actions, including sedation, analgesia, and neuroprotection. Some of these actions (e.g., neuroprotection) may require targets involved in long-term cellular changes. The authors hypothesized that dexmedetomidine increases the expression of active (autophosphorylated) focal adhesion kinase (FAK), a nonreceptor tyrosine kinase playing a pivotal role in cellular plasticity and survival. Therefore, we examined the cellular mechanisms involved in this effect and its sensitivity to oxygen-glucose deprivation (OGD) in rat hippocampal slices. METHODS: The effects of dexmedetomidine on phospho-tyrosine FAK phosphorylation were studied first with or without various pharmacologic agents in normoxic conditions, and second in a model of pharmacologic preconditioning of slices subjected to 30 min of OGD followed by 1 h of reperfusion. FAK phosphorylation and caspase-3 activation were examined by immunoblotting. Neuronal death was assessed by propidium iodide fluorescence. RESULTS: Dexmedetomidine produced a dose-related increase in FAK phosphorylation (187 +/- 4%, mean +/- SD, from basal level, EC50 = 0.2 microm; 95% confidence interval, 0.09-0.5 microm). This effect was stereoselective and was completely blocked by yohimbine and the combination of the cyclic monophosphate permeant analog 8 bromo cyclic monophosphate and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It was mimicked by the protein kinase A inhibitor H 89. In contrast, prazosin and the protein kinase C inhibitors chelerythrine and bisindolylmaleimide I were ineffective. OGD induced a significant increase in immunoreactivity of the cleaved caspase-3 17-kd fragment (417 +/- 22; P < 0.001), a decrease in FAK phosphorylation (78 +/- 12% of control; P < 0.05), and production of significant neuronal death. In OGD conditions, a preconditioning application of dexmedetomidine (0.2 microm, 20-min application, 3 h before anoxia) significantly reduced neuronal death and cleaved caspase-3 expression and significantly attenuated the decrease in phosphorylated FAK content. The dexmedetomidine-induced reduction in caspase-3 expression was significantly decreased by the Src tyrosine kinase inhibitor PP2. CONCLUSION: Dexmedetomidine exhibits a preconditioning effect against ischemic injury in hippocampal slices subjected to OGD. Increase in phosphorylation of FAK via stimulation of alpha2 adrenoceptors and decrease in cleaved caspase-3 expression correlate with dexmedetomidine-induced cell survival.

Effects of anesthetic agents on focal adhesion kinase (pp125FAK) tyrosine phosphorylation in rat hippocampal slices.

BACKGROUND: Tyrosine protein kinase proteins exert a prominent control on signaling pathways and may couple rapid events, such as action potential and neurotransmitter release, to long-lasting changes in synaptic strength and survival. Whether anesthetics modulate tyrosine kinase activity remains unknown. The aim of the current study was therefore to examine the effects of intravenous and volatile anesthetics on the phosphorylation of focal adhesion kinase (ppFAK), a functionally important nonreceptor tyrosine kinase, in the rat hippocampus. METHODS: Phosphorylation of ppFAK was examined in hippocampal slices by immunoblotting with both antiphosphotyrosine and specific anti-ppFAK antibodies. Experiments were performed in the absence (control) or presence of various concentrations of pharmacologic or anesthetic agents or both. RESULTS: Clinically relevant concentrations of thiopental, propofol, etomidate, isoflurane, sevoflurane, and desflurane induced a concentration-related increase in tyrosine phosphorylation. In contrast, ketamine (up to 100 microm) and the nonimmobilizer F6 (1,2-dichlorohexafluorocyclobutane, 25 microm) did not significantly affect ppFAK phosphorylation. The anesthetic-induced increase in ppFAK phosphorylation was blocked by GF 109203X, RO 318220, and chelerythrin (100 microm), three structurally distinct inhibitors of protein kinase C and U 73122 (50 microm), an inhibitor of phospholipase C. The propofol- and isoflurane-induced increase in ppFAK phosphorylation was reversible and showed nonadditivity of effects with phorbol 12-myristate 13-acetate (an activator of protein kinase C, 0.1 microm). In contrast, ketamine (up to 100 microm), MK801 (10 microm, an N-methyl-d-aspartate receptor antagonist), bicuculline (10 microm, a gamma-aminobutyric acid type A receptor antagonist), and dantrolene (30 microm, an inhibitor of the ryanodine receptor) were ineffective in blocking anesthetic-induced activation of tyrosine phosphorylation. CONCLUSION: Except for ketamine, anesthetic agents markedly increase tyrosine phosphorylation of ppFAK in the rat hippocampus, most likely via the phospholipase C-protein kinase C pathway, whereas the nonimmobilizer F6 does not. These results suggest that ppFAK represents a target for anesthetic action in the brain.

Lidocaine increases phosphorylation of focal adhesion kinase in rat hippocampal slices.

We examined the effect of lidocaine on phosphorylation of the tyrosine kinase focal adhesion kinase (PP125FAK) in rat hippocampal slices by immunoblotting with both antiphosphotyrosine and specific anti-PP125FAK antibodies in the presence of tetrodotoxin (1 microM). Lidocaine induced a concentration-related increase in tyrosine phosphorylation of the 125-kDa band corresponding to PP125FAK phosphorylation (EC50 value=0.39+/-0.09 microM, maximal effect=169+/-28% of control, P<0.001). This effect was sensitive to neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK 801, 10 microM) nor the inhibitor of the ryanodine receptor dantrolene (30 microM). In contrast, it was completely blocked by the protein kinase C (PKC) inhibitors chelerythrin, bisindolylmaleimide I (GF 109203X) and bisindolylmaleimide IX (RO-318220, 10 microM). We conclude that lidocaine increases phosphorylation of the tyrosine kinase PP125FAK in the rat hippocampus by a tetrotoxin (TTX)-insensitive mechanism which involves activation of PKC.