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ANESTHETIC PHARMACOLOGY

The Effects of Lidocaine and Bupivacaine on Protein Expression of Cleaved Caspase 3 and Tyrosine Phosphorylation in the Rat Hippocampal Slice

Souhayl Dahmani, MD^*, Danielle Rouelle^*, Pierre Gressens, MD, PhD^*, and Jean Mantz, MD, PhD^*

From the *Department of Anesthesia, Beaujon University Hospital, Assistance Publique des Hôpitaux de Paris and Paris 7 University, Clichy, France; and Institut National de la Santé et de la Recherché Médicale (INSERM U 676), Paris, France.

Address correspondence and reprint requests to S. Dahmani, Department of Anesthesia, Beaujon University Hospital, Assistance Publique des Hôpitaux de Paris and Paris 7 University, 100 Bd du Général Leclerc, 92110 Clichy, France. Address e-mail to souhayl.dahmani{at}bjn.aphp.fr.

	Abstract

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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 µM). 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.

	Introduction

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Rare, but severe, neurologic sequelae have been reported after spinal anesthesia with lidocaine (1). The most devastating adverse event is cauda equina syndrome (2). Clinical neurotoxicity of lidocaine has been considered to be related to the accumulation of high concentrations of this drug in a restricted neuronal environment (3). Although neurotoxic effects of local anesthetics have been reported in numerous histologic studies, the molecular mechanisms involved remain poorly understood. Preferential neuronal target structures, disturbances of cell metabolism, inhibition of -aminobutyric acid-A receptor-mediated currents as well as decreases in neuronal blood flow have been proposed in this regard (4–6).

We have previously shown that pharmacologic concentrations of lidocaine increase the phosphorylation of focal adhesion kinase (FAK, molecular weight: 125 kDa), an important nonreceptor tyrosine kinase playing a pivotal role in coupling rapid events, such as action potential or neurotransmitter release, to long-lasting changes in synaptic plasticity and survival (7). This effect is mediated by protein kinase C and is independent of voltage-operated sodium channels and N-methyl-d-aspartate (NMDA) receptor stimulation (8). Several lines of evidence suggest that the FAK-Src protein complex may play a protective role against neuronal death via antiapoptotic effects involving phoshorylation of extracellular signal regulated kinases, kinases mitogen activated protein (MAP) kinases, and Akt-nuclear factor kB (7). However, proapoptotic actions of FAK mediated via activation of phosphorylation of stress activated protein kinase 2 (P38) and cJunN-terminal kinase JNK MAP kinases have also been reported (7). Therefore, in the present study, we hypothesized that millimolar (neurotoxic) lidocaine concentrations increase FAK phosphorylation, and that this action could lead to activation of caspase 3 in the rat hippocampal slice. Bupivacaine was used for comparative purposes.

	METHODS

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Experiments were performed, after receiving permission from our animal care committee, in an authorized unit and animals were cared for according to the guidelines for the Care Use of Laboratory Animals and guidelines of the National Institute of Health, Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86–23, Bethesda, MD, National Institutes of Health, 1996, pp. 58–9. Forty-eight male Sprague-Dawley rats (Iffa-Credo, L’Arbresle, France) weighing 250 g were housed on a 12:12-h light–dark cycle with food and water ad libitum.

Hippocampal Slice Preparation
Animals were killed by stunning and decapitation. The experimental procedure for slice preparation has been reported in detail elsewhere (8–10). Briefly, hippocampal slices (300 µm thickness each) prepared with a MacIlwain tissue chopper were transferred to polypropylene tubes (three slices per tube) containing 30 mL artificial cerebrospinal fluid (60 min, 37°C). To avoid tyrosine kinase activation at this step of the experiment, Ca²⁺ was omitted from the medium from the dissection phase until the end of incubation. Slices were incubated for 60 min at 37°C with moderate agitation under a humidified atmosphere of O₂/CO₂ 95%/5% (vol/vol) until pharmacological treatments were added together with CaCl₂. Tetrodotoxin (1 µM) was added at the beginning of slice incubation to avoid indirect effects due to neuronal firing. Increasing concentrations of lidocaine (4.3 x 10^–9 to 4.3 x 10^–3 M) and bupivacaine (3.4 x 10^–9 to 3.4 x 10^–3 M) were applied for 5 min in the presence of the phosphatase inhibitor orthovanadate (1 mM). For concentrations of lidocaine (bupivacaine) more than 4.3 x 10^–6 M (3.4 x 10^–6 M, respectively), both 5 and 30 min incubation periods were used. The inhibitor of FAK-Src tyrosine kinase 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2, 10^–5 M, Calbiochem, Nottingham, UK) was administered 1 h before lidocaine and bupivacaine. At the end of the experiments, cerebrospinal fluid was aspirated, slices 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.

Immunoblot Analysis
Protein concentration in the homogenates was determined with a bicinchoninic acid based method, using bovine serum albumin as the standard. Equal amounts of protein (30 µg) were subjected to 6% (wt/vol) polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and transferred electrophoretically to nitrocellulose. For detection of FAK phosphorylation, immunoblot analysis was performed with affinity-purified rabbit antiphosphotyrosine antibodies (monoclonal mouse immunoglobulin [Ig] G, clone 4G10; Euromedex 05-321, Souffelweyersheim, France).

Primary antibodies were labeled with peroxidase-coupled antibodies against rabbit IgG, which were detected by exposure of MP autoradiographic films in the presence of a chemiluminescent reagent (ECL, Amersham, Little Chalfont, UK). The specificity of the immunoreactivity for FAK was assessed by its competition in the presence of 50 µM O-phosphotyrosine. Identification of phosphorylated FAK was performed with a rabbit anti-Y397 FAK phosphospecific antibody (Biosource International, diluted 1:1000) after pooling five to eight independent samples. Identification of total FAK was performed using an anti-FAK antibody directed against the nonphosphorylated residues of the protein (Biosource International, Camarillo, CA; diluted 1:1000). For detection of activated caspase 3, immunoblot analysis was performed with the rabbit polyclonal IgG anti-caspase 3 specific antibodies (diluted 1:2000) detecting both the 32 kDa entire protein and the 17 kDa fragment produced by cleavage of caspase 3 when activated (Upstate Biotechnology, Euromedex, Souffelweyersheim, France). The 17 kDa band was considered cleaved caspase 3 and considered in the statistical analysis. Immunoreactive bands were quantified using a computer-assisted densitometer and normalized to both ß-actin and total FAK expression (quantified by using the specific monoclonal antiactin A5316 antibody (Sigma) ratio; Cohu High Performance CCD camera, Gel Analyst 3.01 pci, Paris, France).

Statistical Analysis
Data were collected from eight independent experiments run in triplicate (three slices per experiment; one rat for three points; total number of rats per condition = 8). Normality of distributions was assessed by the equality of variance test. Concentration–response curves were generated using the GraphPAD 4.0 software (Intuitive Software for Science, San Diego, CA). Statistical analysis was performed using SPSS 12 (Statsoft, Tulsa, OK). The ANOVA test was applied followed by a Bonferroni correction for multiple comparisons. Results are expressed as mean values ± sd. P < 0.05 was considered the threshold for significance.

	RESULTS

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Lidocaine induced a significant concentration-related increase in FAK phosphorylation. Bupivacaine’s effect was biphasic: a concentration-related increase in FAK phosphorylation was observed for concentrations from 1 nM to 100 µM, followed by a decrease when increasing further bupivacaine concentrations (1–4.3 mM, Fig. 1). Concentration–response curves were best fitted by the sigmoidal model for lidocaine, the mean EC₅₀ (95% CI) being 3.3 x 10^–7 M (2–5.2 x 10^–7 M). Bupivacaine-induced FAK phosphorylation was best fitted by the Sine Wave model, the magnitude (95% CI) being 34% (24%–45%) (Fig. 1). Millimolar concentrations of lidocaine induced a greater effect on FAK phosphorylation than clinically equipotent concentrations of bupivacaine (168% (157–183) vs 145%, (133–158) P < 0.001, Fig. 2). Normalizing data to either actin or total FAK did not influence the results (Fig. 2). There was no significant difference in the effects of millimolar lidocaine and bupivacaine concentrations according to the duration of incubation (5 vs 30 min, data not shown). Application of lidocaine (1, 2.3, and 4.3 mM, 30 min) induced a concentration-related increased cleaved caspase 3 expression. In contrast, this was not observed with equipotent bupivacaine concentrations (Fig. 3). The increase in cleaved caspase 3 expression induced by 4.2 x 10^–3 M lidocaine concentration was completely blocked by PP2 (Fig. 3). Micromolar concentrations of both lidocaine and bupivacaine did not affect cleaved caspase 3 expression (Fig. 3).

View larger version (14K): [in this window] [in a new window]   Figure 1. Concentration–response curves for lidocaine and bupivacaine on tyrosine phosphorylation. Tyrosine phosphorylation (mean values ± sd) is expressed as a percentage of control (100%). *P < 0.01 and #P < 0.001 from control.

View larger version (61K): [in this window] [in a new window]   Figure 2. Effects of equipotent millimolar concentrations of lidocaine (L) and bupivacaine (B) on phosphorylation of focal adhesion kinase (FAK). Upper part: Western blots showing (from top to bottom) total FAK expression (phosphorylated plus nonphosphorylated) (tFAK), ß-actin expression (ß-actin), specific phosphorylation of FAK (FAK P279), and total 125 kDa tyrosine phosphorylation (125 kDa-P). **P < 0.01 versus control. ***P < 0.001 versus control.

View larger version (47K): [in this window] [in a new window]   Figure 3. Effects of equipotent millimolar concentrations of lidocaine (L) and bupivacaine (B) and the focal adhesion kinase (FAK) inhibitor PP2 (10 µM) on cleaved caspase 3 expression (P17). **P < 0.01, ***P < 0.001 versus control.

	DISCUSSION

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we have shown that both lidocaine and bupivacaine induce a concentration-related increase in FAK phosphorylation in the rat hippocampal slice. The protein expression of cleaved caspase 3 was increased by millimolar lidocaine, but not bupivacaine concentrations. Lidocaine’s effects on caspase 3 expression were blocked by PP2, an inhibitor of FAK. This suggests that millimolar concentrations of lidocaine may have the potential to induce apoptotic cell death.

Great attention was paid to ensure that the 125 kDa band indeed corresponds to phosphorylated FAK. There was a parallelism in the immunoreactivity labeled by the antiphosphotyrosine and the anti-Y397 FAK phosphospecific antibodies. Also, changes observed for FAK phosphorylation or caspase 3 activation were the consequences of changes in the phosphorylation process per se, and not simply a decrease in total FAK content. Indeed, normalizing the data to either total FAK or to actin, which may be sensitive to neurotoxic conditions, did not significantly affect the results. However, the present study also has limitations. The hippocampal slice may not be the ideal target for investigating the mechanisms of lidocaine toxicity related to spinal anesthesia. However, the choice of the hippocampal slice allows comparison with previous studies (8). Also, millimolar concentrations of lidocaine have been shown to induce caspase 3 activation and apoptotic cell death in a cell line derived from the rat dorsal root ganglion (9).

Both lidocaine and bupivacaine induced a concentration-related increase of FAK phosphorylation for concentrations up to the micromolar range, which confirms and extends our previous findings (8). However, lidocaine-induced FAK phosphorylation reached a plateau when concentrations increased up to 4.3 mM, while bupivacaine’s effect was biphasic. A significant decrease in the magnitude of bupivacaine-induced FAK phosphorylation in comparison with lidocaine was observed for bupivacaine concentrations more than 1 mM. The maximum effect achieved with both drugs was similar to that achieved with NMDA (1 mM) (8). This effect was not mediated by blockade of the voltage-operated sodium channels, since tetrodotoxin, a potent blocker of these channels, was present throughout the experiments. Since NMDA receptor activation is one of the preferential pathways leading to an increase in cytosolic Ca²⁺ concentration and subsequent FAK phosphorylation, it could be speculated that this was the mechanism by which local anesthetics activated the phosphorylation process. However, our findings are not consistent with this hypothesis, since MK 801 was ineffective in this model (8). Also, recent data support the notion that local anesthetics decrease NMDA receptor signaling (10). Finally, we have previously shown that protein kinase C activation plays a key role in lidocaine-induced FAK phosphorylation (8).

The role of FAK phosphorylation in caspase 3 activation by lidocaine, but not bupivacaine, remains to be clarified. Millimolar lidocaine concentrations increased cleaved caspase 3 protein expression. This was blocked by the inhibitor of the FAK-Src tyrosine kinase protein complex PP2. Activation of the Src-family kinases by FAK autophosphorylation is a critical step in allowing phosphorylation on tyrosine residues in the catalytic and carboxyterminal domain of FAK (11,12). PP2 is a highly specific inhibitor or FAK phosphorylation in various tissues, including the brain (13,14). This strongly supports the involvement of FAK in lidocaine-induced caspase 3 expression. Although we actually measured neither necrotic nor apoptotic cell death, it can be speculated that the FAK-Src complex may favor neuronal toxicity and cell death by two different mechanisms: first, NMDA and -aminobutyric acid-A receptor-coupled ionic channels are sensitive to phosphorylation by tyrosine kinases (15). This may induce functional changes in the permeability of these two receptor-coupled channels playing a pivotal role in the development of excitotoxic injury. Alternatively, the FAK-Src tyrosine kinase complex is tightly connected to the MAP kinase cascade, resulting in activation of P38 and JNK MAP kinases (7). Activation of these two phosphoproteins has been demonstrated to be involved in the development of apoptotic cell death (16). However, the lack of effect of bupivacaine millimolar concentrations on caspase 3 expression combined with increased FAK phosphorylation suggests that other mechanisms may explain this difference. Hence, bupivacaine may activate antiapoptotic factors of the Bcl-2 family together with increasing FAK phosphorylation, an effect which may not be shared by lidocaine (17). Alternatively, it cannot be excluded that mechanisms other than FAK phosphorylation account for the difference observed for lidocaine and bupivacaine in the increase of caspase 3 expression.

In conclusion, our results may provide an original working hypothesis to account, at least in part, for the preferential neurotoxic effects reported with lidocaine spinal anesthesia.

	Footnotes

 
Accepted for publication September 25, 2006.

Supported by grants from Paris 7 University and the INSERM.

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