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Dexmedetomidine attenuates lipopolysaccharide-induced proinflammatory response in primary microglia Mian Peng, MD,* Yan-Lin Wang, MD, Cheng-Yao Wang, MD, and Chang Chen, MD Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Wuhan, China
article info
abstract
Article history:
Background: Neuroinflammation mediated by microglia has been implicated in delirium.
Received 25 March 2012
Suppression of microglial activation may therefore contribute to alleviate delirium. It has
Received in revised form
been reported that dexmedetomidine (DEX) has a potent anti-inflammatory property. In
27 April 2012
the present study, we investigated the effects of DEX on the production of proinflammatory
Accepted 10 May 2012
mediators in lipopolysaccharide-stimulated microglia.
Available online 1 June 2012
Materials and methods: The concentrations of DEX were chosen to correspond to 1, 10, and 100 times of clinically relevant concentration (i.e., 1, 10, and 100 ng/mL). The levels of
Keywords:
proinflammatory mediators, such as inducible nitric oxide synthase or nitric oxide, pros-
Dexmedetomidine
taglandin E2, interleukin 1b, and tumor necrosis factor a, were measured.
Nitric oxide
Results: DEX at 1 ng/mL did not affect the production of proinflammatory mediators. DEX at
Inflammation
10 and 100 ng/mL significantly inhibited the release of nitric oxide, prostaglandin E2,
Microglia
interleukin 1b, and tumor necrosis factor a and the expression of inducible nitric oxide synthase messenger RNA. Conclusions: These results suggest that DEX is a potent suppressor of lipopolysaccharideinduced inflammation in activated microglia and may be a potential therapeutic agent for the treatment of intensive care unit delirium. ª 2013 Elsevier Inc. All rights reserved.
1.
Introduction
Delirium is a common neuropsychiatric syndrome that affects elderly intensive care unit (ICU) patients [1,2]. Several lines of studies have indicated that delirium is associated with high morbidity and mortality, increased length of hospital stay, and high rates of institutionalization after discharge [3e6]. Despite these serious effects on outcome, the pathogenesis of delirium is still incompletely understood, but there is growing understanding of the role of neuroinflammation in this condition [7e11]. Microglia are the resident macrophages of the central nervous system and play an important role in
neuroinflammation [12]. Microglia become activated in neuroinflammatory conditions, which initiate an inflammatory cascade in the central nervous system. Overactivated microglia produce excessive amounts of proinflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor a (TNF-a), and interleukin 1b (IL-1b) [13]. These locally produced inflammatory mediators not only modulate further immunologic actions but also affect neuronal function. This effect on the neuronal function has been posited to be the final step that causes clinically manifested cognitive changes (such as delirium) [8,14,15]. Previous studies have demonstrated that activation of microglia is pivotal for the pathogenesis of delirium [7e9]. Therefore,
* Corresponding author. Department of Anesthesiology, Zhongnan Hospital of Wuhan University, 169, Donghu Road, Wuhan 430071, China. Tel.:þ86 15327262471; fax: þ86 2767813256. E-mail address:
[email protected] (M. Peng). 0022-4804/$ e see front matter ª 2013 Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.05.047
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inhibition of excessive microglial activation may be a potential therapeutic target to alleviate the progression of neuroinflammation and delirium [8,16]. Dexmedetomidine (DEX) is a highly selective and potent a2-adrenoreceptor agonist, which was approved by the Food and Drug Administration in 1999 for patients during the first 24 h of mechanical ventilation in the ICU. A number of studies have demonstrated that DEX possesses anti-inflammatory capacity [17e22]. For instance, DEX was reported to reduce endotoxin-induced inflammatory responses in septic rats [21]. DEX was also found to regulate the inflammatory responses induced by lipopolysaccharide (LPS) in murine macrophage [22]. However, nothing is known about the effects of DEX on the inflammatory response in microglia. LPS, a bacterial membrane component, has been shown to be a potent activator of microglia and inducer of brain inflammationeassociated proteins and proinflammatory cytokines in many in vivo and in vitro experimental models [23,24]. In this study, we investigated the effects of DEX on the production of proinflammatory mediators in LPS-stimulated primary microglia. The present study provides information revealing DEX as a potential candidate for use in treatment of ICU delirium.
2.
Materials and methods
2.1.
Cell culture
All animal handling met the approved protocols of Experimental Animal Center Review Board of Wuhan University. Microglia cultures were prepared as previously described [25e27] with some modifications. Briefly, the cerebral cortices of 1-d-old SpragueeDawley rats were minced with a mesh bag (300 mm) and trypsinized. After centrifuging for 10 min at 450 g and for 5 min at 120 g, the tissues were resuspended in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY), containing fetal bovine serum 10% (Gibco), penicillin 100 U/mL, and streptomycin 100 mg/mL (Gibco). Cells were filtered through another mesh bag (53 mm), plated on 75-cm2 culture flasks and kept in DMEM supplemented with fetal bovine serum 10% and the antibiotics in a humidified 5% carbon dioxide atmosphere at 37 C. The medium was changed every 3 d after shaking the flasks to remove neuronal non-glial cells. After incubation for 2 wk, the mixed glial cultures were subcultured into multiwell culture plates. Microglia were isolated from the mixed glial cultures by shaking at 150 rpm for 120 min at 37 C. Detached cells were collected by centrifugation (120 g for 10 min) and seeded at 4 105 cells/cm2. After incubation for 10 min at 37 C, nonadherent or weakly adherent cells were removed by gentle shaking and washed out. The remaining cells were cultured for 24 h and used as microglial cultures. The microglial cultures were verified to be >95% microglia by immunocytochemistry.
2.2.
cells were incubated in serum-free DMEM. In group 2, the cells were treated with 1 mg/mL LPS (from Escherichia coli; Sigma, St Louis, MO). In groups 3e5, the cells were treated with 1, 10, and 100 ng/mL DEX (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h and then stimulated with LPS (1 mg/mL) for 24 h. In the preliminary experiments, we found that the concentrations of 0.5e1.0 mg/mL of LPS are saturating doses for NO release in microglial cells. These concentrations have been used in many other studies measuring LPS-induced NO and TNF-a production in primary microglia cultures [28,29]. Thus, we stimulated microglia with 1 mg/mL of LPS.
2.3.
Cell viability assay
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma) was used to examine the effect of DEX on cell viability. Cell viability was measured based on the formation of blue formazan metabolized from colorless MTT by mitochondrial dehydrogenases, which are active only in live cells. Microglia were plated into 24-well plates at a density of 1 105 cells per well for 24 h and then washed. Cells incubated with various concentrations of DEX for 1 h, with LPS treatment (1 mg/mL) for 24 h and then incubated in 0.5 mg/mL MTT solution. Three hours later, the supernatant was removed, and the formation of formazan was measured at 540 nm using a microplate reader (Model 550; Bio-Rad).
2.4. Reverse transcription and polymerase chain reaction Total RNA was prepared from microglia by using the Trizol reagent (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was reverse transcribed by using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). Polymerase chain reaction (PCR) primers were as follows: inducible nitric oxide synthase (iNOS), sense: 50 —GTG TTC CAC CAG GAG ATG TTG—30 , antisense: 50 — CTC CTG CCC GCT GAG TTC GTC—30 ; b-actin, sense: 50 —TTG TAA CCA ACT GGG ACG ATA TGG—30 , antisense: 50 —GAT CTT GAT CTT CAT GGT GCT AG—30 . The following PCR condition was applied: 24 cycles of denaturation at 95 C for 45 s, annealing at 56 C for 30 s, and extension at 72 C for 1 min. The PCR products were photographed after electrophoresis through agarose gelestained ethidium bromide.
2.5.
NO production
Concentrations of NO in culture supernatants were determined as nitrite, a major product of NO, using Griess reagent (Sigma). Briefly, microglia were pretreated with DEX (1, 10, and 100 ng/mL) for 1 h and then incubated with LPS (1 mg/mL). After 24 h, the supernatants were collected and mixed with the same amount of Griess reagent. Samples were incubated at room temperature for 10 min, and absorbance was subsequently read at 540 nm using a microplate reader (Model 550; Bio-Rad laboratories, Hercules, CA).
Experimental protocols 2.6.
Cells (approximately 1 105 cells/mL) were seeded in six-well plates before being subjected to treatments. Five groups of microglia were subjected to various treatments. In group 1, the
Measurement of PGE2
Microglia were plated at a density of 4 105 cells/mL in a sixwell cell culture plate and incubated with DEX (1, 10, and
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100 ng/mL) in the presence of LPS (1 mg/mL) for 24 h. Following the manufacturer’s instructions, a volume of 100 mL of culture medium supernatant was collected for the determination of PGE2 concentration by enzyme-linked immunosorbent assay (ELISA) (R & D Systems, Minneapolis, MN).
2.7.
Cytokine assays
Cytokine (IL-1b and TNF-a) levels were determined using an ELISA kit (R & D Systems). The absorbance at 450 nm was determined using a microplate reader (Model 550; Bio-Rad).
2.8.
Statistical analysis
Data values represent the means standard error of the mean. Statistical significance was determined using an analysis of variance with Tukey’s multiple comparison post test. A value of P < 0.05 was accepted as statistically significant.
3.
Results
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on inducible nitric oxide synthase messenger RNA (iNOSmRNA) expression and NO release in LPS-stimulated microglia were evaluated by reverse transcriptionePCR and nitrite assay. Microglia were treated with LPS (1 mg/mL) in the presence or absence of DEX for 24 h. A 1-h pretreatment with DEX was carried out before LPS stimulation. DEX pretreatment (1 ng/mL) did not significantly inhibit the LPS-induced iNOSmRNA expression (5.8% 1.1%) and NO production (3.2% 0.3%) compared with the LPS-aloneetreated group (Fig. 2). However, DEX pretreatment (10 and 100 ng/mL) significantly inhibited LPS-induced iNOSmRNA expression by 21.9% 3.5% and 32.2% 9.2%, respectively, and NO production by 31.1% 3.0% and 40.1% 3.1%, respectively, compared with the LPS-aloneetreated group (iNOSmRNA: P < 0.01 and P < 0.001, respectively, and NO: P < 0.001 and P < 0.001, respectively, Fig. 2).
3.3.
Inhibition of LPS-induced PGE2 production by DEX
PGE2, an important inflammatory mediator, was also evaluated for the effects of DEX on its production in LPS-stimulated microglia. As shown in Fig. 3, treatment of microglia with LPS
3.1. No alteration in the viability of microglia treated with DEX and/or LPS Cytotoxic effects of DEX and/or LPS were evaluated by measurement of the viability of microglia using the MTT assay. Compared with serum-free DMEMetreated controls, the viability of microglia was not significantly altered by treatment with DEX and/or LPS (Fig. 1).
3.2. Inhibition of LPS-induced inducible nitric oxide synthase messenger RNA expression and NO release by DEX NO production has been the most widely used representative indicator of microglial activation among many inflammatory mediators. The iNOS expression was generated in activated microglia, mediating synthesis of NO, which were then released into the culture medium. The inhibitory effect of DEX
Fig. 1 e Effect of DEX on cell viability of primary microglia. Cells were treated with the indicated concentration of DEX (1, 10, and 100 ng/mL) for 1 h before LPS (1 mg/mL) treatment for 24 h. Cell viability was assessed using the MTT reduction assay. Each value indicates the mean ± standard error of the mean and is representative of results from three independent experiments.
Fig. 2 e Effects of DEX on iNOSmRNA expression and NO production in LPS-induced primary microglia. (A) The expression of iNOSmRNA was assessed by RT-PCR. The ratio of optical density from iNOS to b-actin bands was calculated. (B) The NO concentration in the culture medium was measured by the Griess reagent. Data represent mean ± standard error of the mean of three independent experiments. ###P < 0.001 versus control group; **P < 0.01, ***P < 0.001 versus LPS group. RT-PCR [ reverse transcriptionePCR.
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Fig. 3 e Effects of PGE2 production in LPS-induced primary microglia. The PGE2 concentration in culture was assayed by an ELISA kit. Data represent mean ± standard error of the mean of three independent experiments. ###P < 0.001 versus control group; ***P < 0.001 versus LPS group.
resulted in a marked increase in PGE2 release in comparison with the serum-free DMEMetreated control after 24 h of exposure to LPS. However, DEX pretreatment (1 ng/mL) did not significantly inhibit the LPS-induced PGE2 production (6.2% 1.9%) compared with the LPS-aloneetreated group (Fig. 3). In contrast, DEX treatment (10 and 100 ng/mL) significantly inhibited LPSmediated PGE2 production by 22.4% 3.4% and 42.1% 3.1%, respectively, compared with the LPS-aloneetreated group (P < 0.001 and P < 0.001, respectively, Fig. 3).
Fig. 4 e Effects of DEX on IL-1b and TNF-a production in LPS-induced primary microglia. (A) The levels of IL-1b and (B) TNF-a in the culture media were measured using ELISA kits. Data represent mean ± standard error of the mean of three independent experiments. ###P < 0.001 versus control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS group.
3.4. Alteration of LPS-induced cytokine production by DEX Proinflammatory cytokines are soluble mediators of inter- and intracellular communications, which occur during inflammatory responses. Release of proinflammatory cytokines (IL-1b and TNF-a) into culture supernatants was determined by ELISA. LPS significantly increased the release of cytokines into the medium. Pretreatment with DEX (10 and 100 ng/mL) significantly attenuated the release of proinflammatory cytokines IL-1b by 12.5% 3.2% and 47.1% 3.3%, respectively, and TNF-a by 10.2% 2.5% and 36.9% 2.6%, respectively, into the medium compared with the LPS-aloneetreated group (IL-1b: P < 0.01 and P < 0.001, respectively, and TNF-a: P < 0.05 and P < 0.001, respectively, Fig. 4), whereas pretreatment with DEX (1 ng/mL) did not significantly suppress the LPS-induced cytokine production (IL-1b: 7.1% 1% and TNF-a: 1% 0.1%) compared with the LPS-aloneetreated group (Fig. 4).
4.
Discussion
The therapeutic plasma concentration range of DEX is 0.3e2 ng/mL [30]. Thus, the concentrations of DEX (1, 10, and 100 ng/mL) correspond to 1, 10, and 100 times clinical plasma concentration. Our present results demonstrate that DEX at clinically relevant concentration (1 ng/mL) did not exert significant effects on regulating the expression of inflammatory molecules in activated primary microglia. In contrast, DEX at concentration higher than clinically relevant ones exhibits
significant inhibitory effects on the production of LPS-induced inflammatory mediators and cytokines, including iNOS or NO, PGE2, IL-1b, and TNF-a in primary microglia. Neuroinflammation is involved in the pathogenesis of delirium [7e11]. Activated microglia play a critical role in the neuroinflammation via releasing various types of inflammatory mediators such as NO, PGE2, IL-1b, and TNF-a. These inflammatory mediators may affect neuronal function and play a key role in cognitive dysfunction during episodes of delirium. For example, proinflammatory cytokine IL-1b can inhibit acetylcholine release and cholinergic-dependent memory function [31,32]. Therefore, inhibition of microglial activation will be a potential therapeutic target to treat delirium. Our data indicated that treatment with DEX (10 and 100 ng/mL) before LPS significantly attenuated the production of inflammatory mediators in activated microglia. Thus, DEX might be a promising therapeutic candidate for ICU delirium. Several clinical studies also demonstrated the benefits of DEX for treatment for ICU delirium. Maldonado et al. [33] reported that 3% of cardiac surgical patients who were randomized to sedation with DEX developed postoperative delirium compared with 50% of those sedated with propofol or midazolam. Riker et al. [34] found that compared with midazolamsedated group, DEX-sedated group had a 24.9% reduction in delirium and a greater mean number of delirium-free days. Pandharipande et al. [35] demonstrated that compared with septic patients who received lorazepam, the DEX septic patients had more delirium-free days. Additionally, sedation
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with DEX, compared with lorazepam, reduced the daily risk of delirium in both septic and nonseptic patients. However, more multicenter, randomized double-blind data are needed to support the benefits of DEX for treatment for ICU delirium. DEX is a potent and highly specific a2-adrenergic agonist. Several studies have demonstrated the anti-inflammatory effects of a2-adrenoreceptor agonists. In vitro, paminoclonidine, an a2-adrenoceptor agonist, suppressed IL-6 production [36], and clonidine suppressed TNF production in monocytes [37]. Moreover, a2-adrenoceptor agonists modulated the LPSinduced production of TNF in macrophages [38]. In recent years, a line of studies demonstrated that DEX possesses antiinflammatory effects, apart from its anesthetic property [17e22]. Qiao et al. [39] and Taniguchi et al. [21] found that DEX may lessen systemic inflammation and increase survival rate in sepsis and endotoxin-induced shock in rats. Can et al. [20] also reported the anti-inflammatory effect of DEX in spinal cord injury rats. Venn et al. [40] and Memis et al. [41] demonstrated that DEX sedation significantly decrease cytokine (IL-1b, TNF-a, and IL-6) production in critically ill patients. Our present study demonstrates that DEX (10 and 100 ng/mL correspond to 10 and 100 times of clinical relevant concentration) inhibited the expression of inflammatory molecules in activated microglia. Similar effects were reported in activated macrophage [22] and cecal ligation and puncture model rats [39]. The utility of DEX in ICU is diverse. The dosage of DEX recommended by the manufacturer for mechanical ventilation is a loading dose of 1 mg/kg over 10 min, followed by a continuous infusion of 0.2e0.7 mg/kg/h for no more than 24 h. The recommended dosage was based on the lack of safety data with high dose and prolonged use. In recent years, a number of studies reported the need for higher dosages of DEX (up to approximately 5e10 times of clinical dosages) than recommended by the manufacturer to achieve adequate sedation [42e45], especially for pediatric patients. The higher dosages of DEX may result in hypotension and bradycardia. However, such dosages of DEX were well tolerated by patients [43,44,46e48]. In addition, Jones et al. [49] found that higher doses of DEX (>0.7 mg/kg/h) did not lead to an increased incidence of hypotension or bradycardia compared with the recommended dosage. On the other hand, animal studies [50] have shown the wide therapeutic windows of DEX for different degrees of sedation and hypnosis. In rats, plasma 50% effective concentration required for the loss of the cornea reflex (24.5 12.3 ng/mL) is >20 fold of that required for the loss of the whisker reflex (1.09 0.10 ng/mL). It seems that the pharmacodynamics actions of DEX are similar for rats and humans [51]. Accordingly, wider dosage range of DEX may be needed to attain different degrees of sedation and hypnosis in human clinical situations. Thus, the use of DEX (10 ng/mL) may be clinically practicable [22]. However, the clinical significance of DEX (100 ng/mL) is negligible because of the impossibility to administrate such a high dosage of DEX to patients. Clinical observations of Maldonado et al. [52,53] demonstrated that normal to low doses of DEX (0.4 mg/kg loading dose followed by 0.2e0.7 mg/kg/h) are sufficient to improve postoperative delirium. These results are in conflict with the potential therapeutic function of DEX (10 ng/mL) for ICU delirium. Many factors may be responsible for the conflicting
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results. First, the study is a small single-center analysis. Second, ICU delirium is multifactorial, not necessarily relying only on neuroinflammation. Finally, the peripheral immune system has a strong effect on the brain. Proinflammatory cytokines, in particular IL-1b and TNF-a, which are induced by surgery and generated in the periphery can be transmitted to the brain and activate microglia [54,55]. Thus, it is possible that the recommended dosages of DEX inhibit indirectly the activation of microglia and improve delirium through suppressing the systemic inflammation and decreasing the productions of plasma proinflammatory cytokines. A series of studies have shown the anti-inflammatory effects of DEX on systemic inflammation [21,22,39]. However, the anti-inflammatory effects of DEX on neuroinflammation (direct inhibition of microglia activation) and systemic inflammation (indirect inhibition of microglia activation) may exist simultaneously. Riker et al. [34] and Pandharipande et al. [35] reported that the high dose and prolonged use of DEX improves ICU delirium. There are limitations in the present study. First, the exact mechanism of anti-inflammatory effect of DEX in activated microglia was not investigated in our study. It is possible that the anti-inflammatory effect is related to its central sympatholytic effects [56,57] and relative stimulation of the cholinergic anti-inflammatory pathway [58,59]. Further studies are required to elucidate the exact mechanism. Second, the magnitude of DEX (10 ng/mL) inhibition observed in the present study was relatively small (up to 10%e31%). This phenomenon may be related to the timing of administration. Taniguchi et al. [60] reported the time-related anti-inflammatory effects of DEX in sepsis rats. They found that early posttreatment of DEX significantly inhibited plasma proinflammatory cytokines production compared with late posttreatment. Therefore, there may be differences in the anti-inflammatory effects of DEX between pretreatment and posttreatment. Further investigations are needed on this point. In conclusion, the present findings indicated that DEX (10 and 100 times clinical relevant plasma concentrations) is a potent suppressor of LPS-induced inflammation in activated microglia and may be a potential therapeutic agent for the treatment of ICU delirium.
Acknowledgment This work was supported by the National Natural Science Foundation of China (309013932).
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