Suppressive effects of levobupivacaine on endotoxin-induced microglial activation

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Suppressive effects of levobupivacaine on endotoxin-induced microglial activation Ya-Hsien Huang, MD,a,b Jiin-Cherng Yen, PhD,b Jie-Jen Lee, MD, PhD,c Jyh-Fei Liao, PhD,b Wen-Jinn Liaw, MD, PhD,d and Chun-Jen Huang, MD, PhDe,f,* a

Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan c Department of Surgery, Mackay Memorial Hospital, Taipei, Taiwan d Department of Anesthesiology, Tri-Service General Hospital, Taipei, Taiwan e Department of Anesthesiology, Buddhist Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan f School of Medicine, Tzu Chi University, Hualien, Taiwan b

article info

abstract

Article history:

Background: We sought to elucidate the effects of levobupivacaine on modulating endotoxin-

Received 15 February 2013

induced upregulation of inflammatory mediators and activation of nuclear factor-kB (NF-kB)

Received in revised form

and mitogen-activated protein kinases (MAPKs) signaling pathways in activated microglia.

17 March 2013

Materials and methods: Confluent murine microglia (BV-2) were treated with endotoxin (lipo-

Accepted 21 March 2013

polysaccharide, 50 ng/mL) or endotoxin plus levobupivacaine (5, 25, or 50 mM) and denoted as

Available online 10 April 2013

the LPS, LPS þ L(5), LPS þ L(25), and LPS þ L(50) groups, respectively. Levobupivacaine was administered immediately after endotoxin. Control groups were run simultaneously.

Keywords:

Results: The concentrations of inflammatory mediators, including macrophage inflamma-

Microglia

tory protein-2 (P ¼ 0.023 and 0.016), tumor necrosis factor-a (P ¼ 0.025 and 0.020), inter-

LPS

leukin (IL)-1b (P ¼ 0.018 and 0.014), IL-6 (P ¼ 0.029 and 0.023), nitric oxide (P ¼ 0.025 and

NF-kB

0.026), and prostaglandin E2 (P ¼ 0.028 and 0.016) of the LPS þ L(25) and LPS þ L(50) groups

MAPK

were significantly lower than those of the LPS group. The concentrations of macrophage

Chemokine

inflammatory protein-2 (P ¼ 0.035), IL-1b (P ¼ 0.024), nitric oxide (P ¼ 0.031), and prosta-

Cytokine

glandin E2 (P ¼ 0.036) but not tumor necrosis factor-a and interleukin-6 of the LPS þ L(5) group were also significantly lower than those of the LPS group. These data revealed that effects of endotoxin on upregulating inflammatory mediators were inhibited by levobupivacaine. Moreover, effects of endotoxin on activating NF-kB, including inhibitor-kB degradation, NF-kB nuclear translocation, and NF-kBeDNA binding, were also inhibited by levobupivacaine. Similarly, effects of endotoxin on activating MAPKs, including extracellular signaleregulated kinase, c-jun N-terminal kinase, and p38 MAPK, were also significantly inhibited by levobupivacaine. Conclusions: Levobupivacaine significantly inhibited endotoxin-induced upregulation of inflammatory mediators and activation of NF-kB and MAPKs signaling pathways in activated microglia. ª 2013 Elsevier Inc. All rights reserved.

Part of the study results have been presented at the Annual Meeting of the American Society of Anesthesiologists, San Francisco, CA, USA, October 13-17, 2007 and Orlando, FL, USA, October 18e22, 2008. * Corresponding author. Department of Anesthesiology, Buddhist Tzu Chi General Hospital, Taipei Branch, 289, Jianguo Rd., Sindian District, New Taipei City 231, Taiwan. Tel.: þ886 2 66289779x2639; fax þ886 2 66289009. E-mail address: [email protected] (C.-J. Huang). 0022-4804/$ e see front matter ª 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.03.074

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1.

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Introduction

Microglia are the major immune components of the central nervous system (CNS) [1]. On exposure to invading pathogens or neuronal debris, microglia can rapidly convert to an activated state and react to destroy the invading pathogens [1]. However, activated microglia can then excessively produce inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), chemokines (e.g., macrophage inflammatory protein-2 [MIP-2]), and cytokines (e.g., tumor necrosis factor-a [TNF-a], interleukin (IL)-1b, and IL-6) [1]. The excessive production of inflammatory mediators from activated microglia can then initiate the process of inflammation and contribute to neural tissue damage in CNS infectious diseases [2,3]. Abundant data have supported the concept that regulation of microglia-mediated inflammation could be a beneficial therapeutic strategy against CNS infectious diseases [3e5]. Local anesthetics exert antinociceptive effects via inhibition of voltage-gated sodium channels and subsequent prevention of cellular depolarization [6]. On top of that, local anesthetics have been reported to possess anti-inflammation capacity [7,8]. This concept was supported by our previous data that bupivacaine, a widely used local anesthetic agent, could significantly inhibit upregulation of inflammatory mediators in endotoxin-stimulated murine macrophages [9]. Levobupivacaine, the pure S ()-enantiomer of bupivacaine, possesses similar local anesthetic potency with reduced cardiac and CNS toxicity compared with its racemic parent bupivacaine [10]. Previous data that levobupivacaine could attenuate bradykinin/substance P-induced vascular flare response in human skin [11] seemed to indicate that levobupivacaine also possesses potent anti-inflammation capacity. This concept was further supported by previous data that rectal administration of levobupivacaine ameliorated colon lesion in rats with experimental colitis [12]. However, direct evidence to confirm the effects of levobupivacaine on mitigating upregulation of inflammatory mediators in activated microglia remain lacking. To elucidate further, we thus conducted this cellular study with the hypothesis that levobupivacaine could attenuate upregulation of inflammatory mediators in endotoxinactivated murine microglia. Moreover, it is well established that endotoxin-induced upregulation of inflammatory mediators is regulated by nuclear factor-kB (NF-kB) and mitogenactivated protein kinases (MAPKs) signaling pathways [13,14]. This study thus also investigated the effects of levobupivacaine on modulating endotoxin-induced activation of NF-kB and MAPKs in microglia.

2.

Materials and methods

2.1.

Cell culture and cell activation protocols

BV-2, an immortalized murine microglial cell line, could readily express functional properties of activated microglia on endotoxin stimulation and thus could serve as a suitable model for inflammation [15]. BV-2 cells were grown and maintained in Dulbecco’s modified Eagle’s medium (Life

Technologies, Grand Island, NY) supplemented with 10% heatinactivated fetal bovine serum and 1% penicillin/streptomycin (Life Technologies), as previously reported [16]. Under a humidified and heated environment (37 C) supplied with a gas mixture of 5% CO2/95% air, cells were plated in 75-cm2 cell culture flasks (Corning, Acton, MA) and split twice a week. Before experiments, cells were plated on six-well dishes (1e2  106 cells per well; Corning). Culture medium was changed every day for routine culture and then 1 h before each experiment. Cells under passage 20 were used for experiments and each treatment was carried out when cells were 80% confluent. Confluent microglia were then stimulated with gram () endotoxin (lipopolysaccharide [LPS] 50 ng/mL, Escherichia coli Serotype 0127:B8; Sigma-Aldrich, St. Louis, MO) to induce expression of the aforementioned pathways [8].

2.2.

Experimental protocols

Microglia were randomized to six groups. Each group contained six culture dishes (n ¼ 6). Two groups were treated with phosphate-buffered saline (PBS, designated as the PBS group; Life Technologies) or LPS (designated as the LPS group) to serve as the negative and positive controls, respectively. Another three groups received LPS plus levobupivacaine (5, 25, or 50 mM; Abbott Laboratories Ltd., Abbott Park, IL) and designated as the LPS þ L(5), LPS þ L(25), or LPS þ L(50) groups, respectively. The last group received levobupivacaine (50 mM, designated as the L(50) group) and served as the control for levobupivacaine. Levobupivacaine was administered immediately after LPS. After reacting with LPS for 24 h or comparable duration in groups without LPS, cell cultures from each group were harvested. The dosages of levobupivacaine were determined according to our preliminary cell viability data that levobupivacaine at the dosage of 5e50 mM posted no significant effects on cell viability in microglia treated with LPS (50 ng/mL).

2.3.

Inflammatory mediator measurements

Freshly harvested culture media were analyzed for the concentrations of MIP-2, TNF-a, IL-1b, IL-6, and PGE2 using enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay kits of MIP-2, TNF-a, IL-1b, IL-6, and PGE2; R&D Systems, Minneapolis, MN). Freshly harvested culture media were also analyzed for the concentrations of stable NO  metabolites, nitrite (NO 2 ) and nitrate (NO3 ), using a colorimetric assay kit (Cayman Chemical, Ann Arbor, MI).

2.4. Analyses of NF-kB and MAPKs activation using immunoblotting assay According to the preliminary inflammatory mediator data, we chose to use only one dosage of levobupivacaine (i.e., 50 mM) for the following experiments. To facilitate the investigation of NF-kB and MAPKs activation, confluent microglia were randomized to receive PBS, levobupivacaine, LPS, or LPS plus levobupivacaine. Each group contained 36 culture dishes (n ¼ 36). Six culture dishes from each group were harvested at 0, 30, 60, 90, 120, and 150 min after LPS administration or compatible durations in groups without LPS.

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Expression of NF-kB and MAPKs were analyzed using immunoblotting assay, as we previously reported [17]. In brief, nuclear and cytosolic extracts were prepared after cell harvesting. After separation by gel electrophoresis, the proteins were transferred to nitrocellulose membranes (BioRad Laboratories, Hercules, CA). For nuclear extracts, the nitrocellulose membranes were incubated overnight at 4 C in primary antibody solution of phosphorylated NF-kB (p-NF-kB p65 [Ser536], 1:500 dilution; Cell Signaling Technology, Inc, Danvers, MA) or Histone H3 (internal standard, 1:500 dilution; Cell Signaling Technology) to facilitate assaying NF-kB nuclear translocation. For cytosolic extracts, the membranes were incubated overnight (4 C) in primary antibody solution of phosphorylated inhibitor-kB (I-kB) (p-I-kBa [Ser32], 1:1000 dilution; Cell Signaling Technology] or actin (internal standard, 1:5000 dilution; Millipore Corporation; Burlington, MA) to facilitate assaying I-kB phosphorylation. For cytosolic extracts, the membranes were also incubated overnight (4 C) in primary antibody solution of phosphorylated extracellular signaleregulated kinase (p-ERK) (1:500 dilution, polyclonal p-ERK1/2 antibody; Santa Cruz Biotechnology, Inc, Santa Cruz, CA), phosphorylated c-jun N-terminal kinase (p-JNK) (1:500 dilution, polyclonal p-JNK1/2 antibody; Santa Cruz Biotechnology, Inc), phosphorylated p38 MAPK (1:200 dilution, polyclonal p-p38 MAPK antibody; Santa Cruz Biotechnology, Inc), or actin (1:5000 dilution; Millipore Corporation) to facilitate assaying MAPKs activation. Horseradish peroxidasee conjugated anti-mouse IgG antibody (Amersham Pharmacia Biotech, Inc, Piscataway, NJ) was used as the secondary antibody. Bound antibody was detected by chemiluminescence (ECL plus kit; Amersham Pharmacia Biotech, Inc) and chemiluminescence film (Hyperfilm; Amersham Pharmacia Biotech, Inc). The protein band densities were quantified using densitometric technology (Scion Image for Windows; Scion Corp, Frederic, MD).

2.5. Immunofluorescent staining for NF-kB nuclear translocation To elucidate further the effects of levobupivacaine on NF-kB nuclear translocation, immunofluorescent staining was performed according to our previous report [17]. In brief, cell cultures were grown on glass coverslips. After reaction with LPS for 90 min, cell cultures were harvested, fixed, permeabilized, and blocked. Coverslips were incubated 30 min in primary antibody solution of phosphorylated NF-kB (p-NF-kB p65 [Ser536], 1:100 dilution; Cell Signaling Technology). After washing, cells were incubated with fluorescent rhodamine isothiocyanateeconjugated secondary antibody (Jackson ImmunoResearch, Inc, West Grove, PA) for 30 min. Nuclear counterstaining with diamidino-2-phenylindole (Pierce Biotechnology, Rockford, IL) was then performed and the cells were imaged using a confocal microscope (TCS SP5 AOBS; Leica Microsystems CMS GmbH, Mannheim, Germany).

2.6. Electrophoretic mobility shift assay for NF-kBeDNA binding NF-kB activation is also evidenced by increases in NF-kBeDNA binding [18]. To facilitate investigation, cell cultures were

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grown in culture dishes. After reaction with LPS for 90 min, cell cultures were harvested and nuclear extracts were prepared as aforementioned. Electrophoretic mobility shift assay was then performed using a chemiluminescence electrophoretic mobility shift assay kit (Panomics, Inc, Fremont, CA), as we have previously reported [17].

2.7.

Statistical analysis

Data of inflammatory mediators were analyzed using oneway analysis of variance (ANOVA) with Tukey post hoc test to determine the between-group difference. Immunoblotting assay data of NF-kB, I-kB, ERK, JNK, and p38 MAPK were analyzed with repeated-measures ANOVA with Bonferroni corrections to examine the within-group (time) effect, between-group effect, and the group by time interaction effect. Data were presented as means and standard deviations. The significance level was set at 0.05. A commercial software package (SPSS 11.5 for Windows; SPSS Science, Chicago, IL) was used for data analysis.

3.

Results

3.1.

Inflammatory mediators

The concentrations of MIP-2, TNF-a, IL-1b, IL-6, NO, and PGE2 of the PBS and L(50) groups were low (data not shown). As expected, the concentrations of MIP-2, TNF-a, IL-1b, IL-6, NO, and PGE2 of the LPS group were significantly higher than those of the PBS group (all P < 0.001). In contrast, the MIP-2, TNF-a, IL-1b, IL-6, NO, and PGE2 concentrations of the LPS þ L(25) and LPS þ L(50) groups were significantly lower than those of the LPS group (MIP-2: P ¼ 0.023 and 0.016; TNF-a: P ¼ 0.025 and 0.020; IL-1b: P ¼ 0.018 and 0.014; IL-6: P ¼ 0.029 and 0.023; NO: P ¼ 0.025 and 0.026; and PGE2: P ¼ 0.028 and 0.016, Fig. 1). The concentrations of MIP-2, IL-6, NO, and PGE2, but not TNF-a and IL-1b, of the LPS þ L(5) group were also significantly lower than those of the LPS group (MIP-2: P ¼ 0.035; IL-1b: P ¼ 0.024; NO: P ¼ 0.031; and PGE2: P ¼ 0.036; Fig. 1).

3.2.

NF-kB activation

Repeated-measures ANOVA revealed that the trend of NF-kB activation, including NF-kB nuclear translocation and I-kB degradation, was significantly different among these four groups (P < 0.001). The concentrations of p-NF-kB p65 in nuclear extracts and p-I-kBa in cytosolic extracts of the PBS and L(50) groups were low throughout the experiment (data not shown). As expected, the concentrations of p-NF-kB p65 in nuclear extracts and p-I-kBa in cytosolic extracts in cell cultures harvested at 30, 60, 90, 120, and 150 min after LPS administration of the LPS group were significantly higher than those of the PBS group (all P < 0.001). Moreover, the concentrations of p-NF-kB p65 in nuclear extracts of the LPS þ L(50) group harvested at 90, 120, and 150 min after LPS were significantly lower than those of the LPS group (P ¼ 0.028, 0.024, and 0.008; Fig. 2A). Similarly, the concentrations of p-I-kBa in cytosolic extracts of the LPS þ L(50) group harvested at 60, 90, 120, and 150 min after LPS were significantly lower

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Fig. 1 e The concentrations of MIP-2, TNF-a, IL-1b, IL-6, NO, and PGE2 in endotoxin-activated microglia. LPS: the lipopolysaccharide (50 ng/mL) group. LPS D L(5): the LPS plus levobupivacaine (5 mM) group. LPS D L(25): the LPS plus levobupivacaine (25 mM) group. LPS D L(50): the LPS plus levobupivacaine (50 mM) group. Data were derived from six culture dishes from each group and expressed as means ± standard deviations. #P < 0.05 versus the LPS group.

than those of the LPS group (P ¼ 0.031, 0.022, 0.011, and 0.013, respectively; Fig. 2A). NF-kB nuclear translocation was also evaluated by immunofluorescent staining. Data of the immunofluorescent staining revealed that the fluorescence intensities of the PBS and L(50) groups were low and the fluorescence intensity of the LPS group was stronger than that of the PBS group (data not shown). Moreover, the fluorescence intensity of the LPS þ L(50) group was weaker than that of the LPS group (Fig. 2B). The NF-kBeDNA binding of the PBS and L(50) groups were also low (data not shown). As expected, the NF-kBeDNA binding of the LPS group was significantly higher than that of the PBS group (P < 0.001, data not shown). Similarly, the NF-kBeDNA binding of the LPS þ L(50) group was significantly lower than that of the LPS group (P ¼ 0.025, Fig. 2C).

were significantly higher than those of the PBS group (all P < 0.001). Moreover, the p-ERK protein concentrations of the LPS þ L(50) group in cell cultures harvested at 30, 60, 90, 120, and 150 min after LPS were significantly lower than those of the LPS group (P ¼ 0.027, 0.013, 0.033, 0.031, and 0.024, respectively; Fig. 3). The p-JNK protein concentrations of the LPS þ L(50) group in cell cultures harvested at 30, 60, 90, 120, and 150 min after LPS were also significantly lower than those of the LPS group (P ¼ 0.003, 0.034, 0.016, 0.029, and 0.030, respectively; Fig. 3). Similarly, the p-p38 MAPK protein concentrations of the LPS þ L(50) group in cell cultures harvested at 90, 120, and 150 min after LPS were significantly lower than those of the LPS group (P ¼ 0.013, 0.021, and 0.002, respectively; Fig. 3).

3.3.

4.

MAPKs activation

Repeated-measures ANOVA also revealed that the trend of MAPKs activation, including ERK, JNK, and p38 MAPK activation, was significantly different among these four groups (P < 0.001). The p-ERK, p-JNK, and p-p38 MAPK concentrations of the PBS and L(50) groups were low throughout the experiment (data not shown). The p-ERK, p-JNK, and p-p38 MAPK protein concentrations of the LPS group in cell cultures harvested at 30, 60, 90, 120, and 150 min after LPS administration

Discussion

Data from the present study clearly demonstrated that levobupivacaine significantly inhibited endotoxin-induced upregulation of inflammatory mediators, including chemokine (e.g., MIP-2), cytokines (e.g., TNF-a, IL-1b, and IL-6), NO, and PGE2, in activated microglia. These data confirmed our hypothesis. Moreover, these data in concert with those from previous studies [11,12] confirmed the potent antiinflammation capacity of levobupivacaine. Regulation of

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Fig. 2 e (A) Representative gel photography and densitometric analysis data of phosphorylated NF-kB p65 (p-NF-kB p65) protein concentrations in the nuclear extracts and phosphorylated inhibitor-kBa (I-kBa) protein concentrations in the cytosolic extracts of endotoxin-activated microglia using immunoblotting assay. The p-NF-kB p65 protein concentrations were normalized by Histone-H3. The p-I-kBa protein concentrations were normalized by actin. (B) Representative findings of immunofluorescent staining assay for p-NF-kB p65 in endotoxin-activated microglia (i.e., NF-kB nuclear translocation). The p-NF-kB p65 protein was stained with fluorescent rhodamine isothiocyanateeconjugated antibody. Nuclei were counterstained with diamidino-2-phenylindole. The cells were imaged using a confocal microscope. (C) Representative gel photography and densitometric analysis data of the NF-kBeDNA binding in the nuclear extracts of endotoxin-activated microglia using chemiluminescence electrophoretic mobility shift assay. LPS: the lipopolysaccharide (50 ng/mL) group. LPS D L(50): the LPS plus levobupivacaine (50 mM) group. Data were derived from six culture dishes from each group and expressed as means ± standard deviations. #P < 0.05 the LPS D L(50) group versus the LPS group. (Color version of figure is available online.)

microglia-mediated inflammation is an important therapeutic strategy against CNS infectious diseases [3e5]. In line with this notion, we thus speculate that incorporating levobupivacaine as a therapeutic adjunct might be beneficial and could be a novel therapeutic strategy against CNS infectious diseases. As aforementioned, endotoxin-induced upregulation of inflammatory mediators is regulated by NF-kB and MAPKs [13,14]. We thus also investigated the effects of levobupivacaine on regulating expression of NF-kB and MAPKs in activated microglia. Data from the present study confirmed that levobupivacaine significantly mitigated endotoxin-induced NF-kB activation, as our data demonstrated that the levels of endotoxin-induced I-kB degradation, NF-kB nuclear translocation, and NF-kBeDNA binding in microglia treated with endotoxin and levobupivacaine were significantly lower than

those in microglia treated with endotoxin alone. Our data that the protein concentrations of p-ERK, p-JNK, and p-p38 MAPK in microglia treated with endotoxin and levobupivacaine were significantly lower than those in microglia treated with endotoxin alone also confirmed the effects of levobupivacaine on mitigating endotoxin-induced activation of MAPKs. Judging from these data, we believe that levobupivacaine may very likely act through regulating the upstream transcriptional factors NF-kB and MAPKs to modulate expression of inflammatory mediators in activated microglia. Although data from the present study confirmed the effects of levobupivacaine on inhibiting endotoxin-induced activation of NF-kB and MAPKs in microglia, the underlying mechanisms remain unstudied. Binding of endotoxin to inflammatory cells and subsequent activation of the membrane surface

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Fig. 3 e Representative gel photography and densitometric analysis data of protein concentrations of activated MAPKs, including p-ERK, p-JNK, and p-p38 MAPK, in the cytosolic extracts of endotoxin-activated microglia using immunoblotting assay. The MAPKs protein concentrations were normalized by actin. LPS: the lipopolysaccharide (50 ng/mL) group. LPS D L(50): the LPS plus levobupivacaine (50 mM) group. Data were derived from six culture dishes from each group and expressed as means ± standard deviations. #P < 0.05 the LPS D L(50) group versus the LPS group.

receptor toll-like receptor 4 (TLR-4) is essential for NF-kB and MAPKs activation [19e23]. For endotoxin binding, LPS will initially interact with LPS-binding protein and then be transferred to cluster of differention-14 (CD14), a membraneanchor protein that mediates the recognition of endotoxin [19,24]. After binding, endotoxin induces a physical proximity of CD14 and TLR-4 that allows CD14 to present endotoxin to TLR-4, which leads to the activation of intracellular signaling pathways [19,25]. In addition, myeloid differential-2 (MD-2), an extracellular protein associated with extracellular domain of TLR-4, is also required for the activation of TLR-4 by endotoxin [19,26]. Previous data also indicated that the activity of LPS depends largely on lipid A (a primary structure of LPS), as binding of LPS to CD14/TLR-4/MD-2 complex requires a high density of negative charge in the lipid A backbone and only conical-shaped, but not cylindrical-shaped, lipid A molecules exhibited biological activity [27]. Previous data also indicated that the shape of the lipid A molecules could be converted from cylindrical to conical and this conversion may involve activation of the high-conductance Ca2þ-activated potassium channels induced by endotoxin [27,28]. The effects of levobupivacaine on modulating endotoxin binding and the subsequent activation of CD14/TLR-4/MD-2 complex remain unstudied. The effects of levobupivacaine on the Ca2þactivated potassium channels remain unstudied, either.

However, judging from recent data that bupivacaine could inhibit the Ca2þ-activated potassium channels [29], we speculate that levobupivacaine might post similar effects on modulating the Ca2þ-activated potassium channels. If so, then it is likely that levobupivacaine could enhance the conversion of conical-shaped lipid A molecules to cylindrical-shaped lipid A molecules (i.e., neutralize LPS) and subsequently inhibit endotoxin binding and CD14/TLR-4/MD-2 complex activation. More studies are needed before further conclusion scan be drawn. Data from the present study illustrated that levobupivacaine at the dosage of 5 mM could attenuate the effects of endotoxin on upregulating MIP-2, IL-1b, NO, and PGE2, but not TNF-a and IL-6, in activated microglia. Our data further illustrated that levobupivacaine at the dosages of 25 and 50 mM could inhibit the upregulation of all inflammatory mediators investigated in this study. These data indicate that the antiinflammation capacity of levobupivacaine is dose dependent. As aforementioned, regulation of microglia-mediated inflammation could be beneficial against CNS infectious diseases [3e5]. Judging from our data, we thus speculate that 25e50 mM levobupivacaine can be an effective therapeutic adjunct against CNS infection diseases. Future in vivo studies are needed before we can conclude on our speculation. However, if future in vivo data do confirm our concept, one

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major drawback of this application will be the possible sensory and motor blockade caused by intrathecal levobupivacaine. The clinical dosage of levobupivacaine for neuraxial anesthesia (e.g., intrathecal administration of 0.5% levobupivacaine) is approximately 17.4 mM [30,31]. As the dosage range for levobupivacaine to cause significant sensory and motor blockade may be 350e700 times higher than the dosage range for levobupivacaine to exert significant antiinflammation effect, it is possible that we can develop effective dosage ranges of intrathecal levobupivacaine against CNS infection disease without causing significant sensory and/or motor blockade. Although this study used endotoxin-induced activation of microglia model to confirm the anti-inflammation capacity of levobupivacaine, the clinical implication of our data may not be limited only to CNS infectious diseases. Similar to CNS infectious disease, microglia activation and subsequent inflammatory mediators overproduction also play essential roles in initiating the process of inflammation and contribute to the progress of several neurodegenerative diseases, including Alzheimer disease, Parkinson disease, multiple sclerosis, and so forth [3,32]. Similar mechanism is also observed in neuropathic pain [33,34]. As levobupivacaine possesses potent anti-inflammation capacity, we thus speculate that levobupivacaine can also be an effective therapeutic adjunct against neurodegenerative diseases and/or neuropathic pain. Certain study limitations do exist. First, only very few data are available regarding the anti-inflammation effects of levobupivacaine [11,12]. As this study used only one cell line to facilitate investigation, the question of whether levobupivacaine could post similar effects in other cell lines and/or in intact animals remains unstudied. Second, the question of whether the other local anesthetics can exert similar antiinflammation effects in this regard remains unanswered. However, our previous data confirmed the anti-inflammation capacity of bupivacaine and lidocaine [8,9]. In addition, previous data also demonstrated the anti-inflammation capacity of ropivacaine [11]. Based on these data, we speculate that these above-mentioned local anesthetics may exert antiinflammation effects similar to those of levobupivacaine observed in this study. Future studies are needed to conclude on our speculation. In summary, data from the present study demonstrated that levobupivacaine could significantly inhibit endotoxininduced upregulation of inflammatory mediators and activation of NF-kB and MAPKs signaling pathways in activated microglia.

Acknowledgment The authors express their appreciations to Professors Yen-Jen Sung and Huey-Jen Tsay from National Yang-Ming University, Taipei, Taiwan for their generosity in supplying BV-2 cells for this study. This work was supported by a grant from the National Science Council, Taiwan (NSC 96-2314-B-195-004-MY3) awarded to C.-J.H.

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references

[1] Perry VH, Gordon S. Macrophages and microglia in the nervous system. Trends Neurosci 1988;11:273. [2] Matsumoto Y, Ohmori K, Fujiwara M. Microglial and astroglial reactions to inflammatory lesions of experimental autoimmune encephalomyelitis in the rat central nervous system. J Neuroimmunol 1992;37:23. [3] Dheen ST, Kaur C, Ling EA. Microglial activation and its implications in the brain diseases. Curr Med Chem 2007;14: 1189. [4] Chang Y, Lee JJ, Hsieh CY, Hsiao G, Chou DS, Sheu JR. Inhibitory effects of ketamine on lipopolysaccharideinduced microglial activation. Mediators Inflamm 2009;2009: 705379. [5] Castano A, Herrera AJ, Cano J, Machado A. The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone, and not mimicked by rh-TNF-alpha, IL-1beta and IFN-gamma. J Neurochem 2002;81:150. [6] Butterworth JF 4th, Strichartz GR. Molecular mechanisms of local anesthesia: a review. Anesthesiology 1990;72: 711. [7] Hollmann MW, Durieux ME. Local anesthetics and the inflammatory response: a new therapeutic indication? Anesthesiology 2000;93:858. [8] Huang YH, Tsai PS, Kai YF, Yang CH, Huang CJ. Lidocaine inhibition of inducible nitric oxide synthase and cationic amino acid transporter-2 transcription in activated murine macrophages may involve voltage-sensitive Naþ channel. Anesth Analg 2006;102:1739. [9] Huang YH, Tsai PS, Huang CJ. Bupivacaine inhibits COX-2 expression, PGE2, and cytokine production in endotoxinactivated macrophages. Acta Anaesthesiol Scand 2008;52: 530. [10] Burlacu CL, Buggy DJ. Update on local anesthetics: focus on levobupivacaine. Ther Clin Risk Manag 2008;4:381. [11] Dias MP, Newton DJ, McLeod GA, Khan F, Belch JJ. The inhibitory effects of local anaesthetics on the vascular flare responses to bradykinin and substance P in human skin. Anaesthesia 2008;63:151. [12] Duman U, Yilmazlar A, Ozturk E, Aker S, Sarandol E, Yilmazlar T. Anti-inflammatory efficiency of levobupivacaine in an experimental colitis model. World J Gastroenterol 2010;16:2537. [13] Christman JW, Lancaster LH, Blackwell TS. Nuclear factor kappa B: a pivotal role in the systemic inflammatory response syndrome and new target for therapy. Intensive Care Med 1998;24:1131. [14] Chen C, Chen YH, Lin WW. Involvement of p38 mitogenactivated protein kinase in lipopolysaccharide-induced iNOS and COX-2 expression in J774 macrophages. Immunology 1999;97:124. [15] Henn A, Lund S, Hedtjarn M, Schrattenholz A, Porzgen P, Leist M. The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX 2009; 26:83. [16] Long TC, Tajuba J, Sama P, et al. Nanosize titanium dioxide stimulates reactive oxygen species in brain microglia and damages neurons in vitro. Environ Health Perspect 2007;115: 1631. [17] Chen CP, Tsai PS, Huang CJ. Antiinflammation effect of human placental multipotent mesenchymal stromal cells is mediated by prostaglandin E2 via a myeloid differentiation primary response gene 88-dependent pathway. Anesthesiology 2012;117:568.

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[18] Nemeth ZH, Deitch EA, Lu Q, Szabo C, Hasko G. NHE blockade inhibits chemokine production and NF-kappaB activation in immunostimulated endothelial cells. Am J Physiol Cell Physiol 2002;283:C396. [19] Heumann D, Roger T. Initial responses to endotoxins and Gram-negative bacteria. Clin Chim Acta 2002;323:59. [20] Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 1997; 17:3. [21] Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 1993;268:14553. [22] Asehnoune K, Strassheim D, Mitra S, Kim JY, Abraham E. Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappa B. J Immunol 2004;172: 2522. [23] Chi H, Barry SP, Roth RJ, et al. Dynamic regulation of proand anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses. Proc Natl Acad Sci USA 2006;103:2274. [24] Fenton MJ, Golenbock DT. LPS-binding proteins and receptors. J Leukoc Biol 1998;64:25. [25] Jiang Q, Akashi S, Miyake K, Petty HR. Lipopolysaccharide induces physical proximity between CD14 and toll-like receptor 4 (TLR4) prior to nuclear translocation of NF-kappa B. J Immunol 2000;165:3541. [26] Nagai Y, Akashi S, Nagafuku M, et al. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 2002;3:667.

[27] Schromm AB, Brandenburg K, Loppnow H, et al. Biological activities of lipopolysaccharides are determined by the shape of their lipid A portion. Eur J Biochem 2000;267:2008. [28] Seydel U, Scheel O, Muller M, Brandenburg K, Blunck R. A Kþ channel is involved in LPS signaling. J Endotoxin Res 2001;7: 243. [29] Martin P, Enrique N, Palomo AR, Rebolledo A, Milesi V. Bupivacaine inhibits large conductance, voltage- and Ca2þactivated Kþ channels in human umbilical artery smooth muscle cells. Channels (Austin) 2012;6:174. [30] Shim HS, Ok SH, Lee SH, Kwon SC, Sohn JT. Protein kinases participate in the contraction in response to levobupivacaine in the rat aorta. Eur J Pharmacol 2012;677:131. [31] Costello TG, Cormack JR, Mather LE, LaFerlita B, Murphy MA, Harris K. Plasma levobupivacaine concentrations following scalp block in patients undergoing awake craniotomy. Br J Anaesth 2005;94:848. [32] Itagaki S, McGeer PL, Akiyama H, Zhu S, Selkoe D. Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease. J Neuroimmunol 1989;24: 173. [33] Graeber MB, Christie MJ. Multiple mechanisms of microglia: a gatekeeper’s contribution to pain states. Exp Neurol 2012; 234:256. [34] Tenorio G, Kulkarni A, Kerr BJ. Resident glial cell activation in response to perispinal inflammation leads to acute changes in nociceptive sensitivity: implications for the generation of neuropathic pain. Pain 2013;154:71.