Departments of 1 Anesthesiology and Critical Care Medicine and 2 Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, 39 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan
* Corresponding author. Present address: Department of Anesthesiology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi 409-3898, Japan. E-mail: tando{at}yamanashi.ac.jp
Accepted for publication August 16, 2005.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Glial cells were stimulated with LPS in the absence and presence of various concentrations of ketamine (301000 µM) or propofol (30 and 300 µM). Nitric oxide released into the culture media was determined by measuring nitrite using the Griess reaction, and concentrations of TNF- and PGE2 were measured by enzyme-linked immunosorbent assay (ELISA).
Results. Ketamine reduced LPS-induced TNF- production without significant inhibition of nitrite release in mixed glial cells, astrocyte cultures and microglial cultures. Ketamine also inhibited LPS-induced production of PGE2 in astrocyte cultures. In contrast, propofol had no effect on LPS-induced nitrite or TNF-
production in mixed glial cells.
Conclusions. The data demonstrate that ketamine inhibited some of the inflammatory responses of both astrocytes and microglial cells treated with LPS without causing major change in nitric oxide release. Propofol had no effect on the production of nitric oxide or TNF- from LPS-stimulated glial cells.
Keywords: anaesthetics i.v., ketamine ; anaesthetics i.v., propofol ; immune response ; pharmacology, ketamine ; pharmacology, propofol
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ketamine shows anti-inflammatory actions in various immune cells, such as macrophages and peripheral leucocytes, stimulated with lipopolysaccharide (LPS) in vitro and in vivo 9 11. There have been several studies of the effects of propofol (2,6-diisopropylphenol) on cytokine release from LPS-stimulated immune cells; however, conflicting results demonstrating both inhibition and augmentation have been reported.12 14 The effects of these agents on the inflammatory responses of native glial cells have yet to be clarified. It is known that there are differences in the regulation of LPS-induced inflammatory responses between macrophages and glial cells.15 17 Nitric oxide and tumour necrosis factor- (TNF-
) play key roles in acute and chronic neurodegenerative processes and their LPS-stimulated production in macrophages and leucocytes has been shown to be suppressed by ketamine.9 11 We measured changes in these mediators to compare the effects of ketamine on the inflammatory responses in glial and other immune cells. Prostaglandins released from glial cells are shown to be involved in the pathogenesis of neurological disorders related to inflammation.18 We chose prostaglandin E2 (PGE2) because this molecule plays an important role in pathological pain at the spinal level19 and mediates gliaglia and glianeuron communication in various pathological conditions, including inflammation, by stimulating glutamate release from astrocytes.20 In this study, we investigated the effects of ketamine and propofol on LPS-induced production of nitric oxide, TNF-
and PGE2 in primary cultures of rat glial cells in vitro.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents
Racemic ketamine hydrochloride, LPS (serotype 055B5), D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) and aminoguanidine were obtained from Sigma (St Louis, MO). Propofol was purchased from Aldrich (Oakville, Canada) and was dissolved in dimethyl sulphoxide (DMSO) shortly before application to make a 200 mM solution. Dulbecco's modified Eagle's medium (DMEM), L-15 medium and trypsin were purchased from Gibco (Grand Island, NY), and fetal bovine serum (FBS) was obtained from Wako (Osaka, Japan).
Primary culture of mixed glial cells
Cultures were prepared from whole brains of 2-day-old Wistar rats using the procedure described previously21 22 with some modifications. The meninges and blood vessels were carefully removed, and the tissue was minced with a mesh bag (300 µm) and trypsinized (trypsinEDTA 2.5% and DNase 0.1% in L-15 medium). After centrifuging for 10 min at 450g and for 5 min at 120 g, the tissues were resuspended in DMEM containing FBS 10%, penicillin and 100 U ml1 and streptomycin 100 µg ml1. Cells were filtered through another mesh bag (53 µm), plated on 75 cm2 culture flasks and kept in DMEM supplemented with FBS 10% and the antibiotics in a humidified 5% carbon dioxide atmosphere at 37°C. The medium was changed every 3 days after shaking the flasks to remove neuronal non-glial cells. After 1213 days in vitro, cultures were subcultured into multi-well culture plates and used after 2 days as mixed glial cells.
Secondary cultures of astrocytes
Mixed glial cultures grown for 12 or 13 days in 75 cm2 flasks were shaken at 150 r.p.m. for 120 min at 37°C on a gyratory shaker. The remaining source cultures were dissociated using trypsin and then collected by centrifuging (120 g for 5 min). The cells were seeded onto 24-well culture plates at 2x105 cells cm2 and cultured for 24 h before being used as astrocyte cultures.
Secondary cultures of microglia
Microglial cells were harvested from mixed glial cultures in 75 cm2 flasks by shaking at 150 r.p.m. for 120 min at 37°C. Detached cells were collected by centrifugation (120 g for 10 min) and seeded at 4 x105 cells cm2. After incubation for 10 min at 37°C, non-adherent 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.
Immunocytochemistry
The cultures were fixed with paraformaldehyde 4% in phosphate-buffered saline (PBS) 0.1 M for 2 h and rinsed three times with PBS 0.1 M. Non-specific binding was blocked with 10% bovine serum albumin (BSA) for 5 h at room temperature. Subsequently, cultures were incubated with mouse monoclonal antibodies (1:200 dilution) to the specific markers of microglia (OX-42 against CD 11b surface antigen) or astrocytes (glial fibrillary acidic protein [GFAP]) in the blocking buffer for 24 h at 4°C. After washing three times with PBS, the cells were incubated with the secondary antibody (Alexa Fluor 594 goat anti-mouse IgG, 1:1000 dilution [Molecular Probes, Eugene, OR]) for 2 h at room temperature and rinsed. The preparations were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and examined using an inverted microscope equipped with fluorescence optics. We calculated the percentage of positively immunostained cells (in 200 cells) in each of three separate culture preparations obtained on different days.
Treatment of cultures
Glial cells were preincubated with ketamine (30, 100, 300 or 1000 µM) or propofol (30 or 300 µM) for 15 min, and then LPS was added at final concentrations of 0.510 µg ml1 for 24 h in the continuous presence of ketamine or propofol. In the preliminary experiments we found that the concentrations of 0.51.0 µg ml1 of LPS are saturating doses for nitric oxide release in mixed glial cells and astrocytes. These concentrations have been employed in many other studies measuring LPS-induced nitric oxide and TNF- production in primary glial cultures.23 24 Thus we stimulated mixed glial cells and astrocytes with 0.5 or 1.0 µg ml1 of LPS. For microglia, we used 10 µg ml1 of LPS because we found that 1.0 µg ml1 was not sufficient to produce distinct increases in nitrite. In some experiments, cells were pretreated with aminoguanidine, a blocker for inducible nitric oxide synthetase (iNOS), or D-AP5, an N-methyl-D-asparate (NMDA) receptor antagonist,25 before stimulation with LPS. The culture media were collected after 24 h and centrifuged, and the supernatants were subjected to the assays described below.
Measurement of released nitrite, TNF- and PGE2
The amount of nitric oxide released from glial cells was determined by assaying nitrite, a relatively stable metabolite of nitric oxide. Nitrite concentrations in the supernatants were measured using the Griess reaction as described previously.26 The optical density of assay samples was measured spectrophotometrically at 570 nm. Nitrite concentrations were determined from a standard curve constructed using the known concentrations of sodium nitrite. The concentrations of TNF- and PGE2 in the supernatants of the culture media were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MO) according to the manufacturer's instructions. The measurement sensitivities were 125 pmol for nitrite, 5 pg ml1 for TNF-
and 36.2 pg ml1 for PGE2.
Assessment of the number of viable cells
The number of viable cells in each well after LPS treatment was assessed using a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.27 The optical density of the reaction media was determined at 550 nm.
Statistics
All samples were assayed in duplicate and the values were averaged. Data are expressed as median (IQR). The MannWhitney test was used for comparison between two groups. Multiple comparisons against a control group not treated with anaesthetic were made using the KruskalWallis test followed by the MannWhitney test for post hoc testing. A P-value <0.05 was considered significant. Concentrationinhibition curves were fitted to the Hill equation
![]() |
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although ketamine has been shown to exert anti-inflammatory actions on a variety of immune cells, the exact mechanisms responsible for these actions are not well understood.9 11 We have shown that high doses of ketamine caused no significant changes in the number of viable cells estimated by MTT reduction assay. This finding excluded the possibility that the release of the inflammatory mediators is inhibited by the cytotoxic actions of ketamine. Our finding that ketamine reduced production of TNF- and PGE2 without affecting nitric oxide release is intriguing but inconsistent with earlier studies reporting inhibitory effects of ketamine on the release of both TNF-
and nitric oxide from a macrophage-like cell line and alveolar macrophages in response to LPS.10 31 The mechanisms for the differential effects on nitric oxide release from different cells remain to be clarified. It is known that LPS stimulation causes activation and nuclear translocation of nuclear factor
B (NF-
B) and activator protein 1 (AP-1), leading to transcriptional activation of proinflammatory genes, such as those encoding iNOS, TNF-
and cyclooxygenase-2, in glial cells as well as macrophages.2 However, a number of studies have revealed differences in the regulation of LPS-induced expression of iNOS and inflammatory cytokines between macrophages and glial cells. Certain intracellular signalling molecules, such as cyclic AMP and protein phosphatases, are known to regulate LPS-stimulated iNOS expression in opposite directions in macrophages and astrocytes.15 16 The mitogen-activated protein kinases ERK-1 and ERK-2 have different roles in LPS-induced signalling in macrophages and microglia.17 These cell-type-specific signalling pathways are likely to contribute to the different effects of ketamine observed in macrophages and glial cells. In another study which investigated the effects of ketamine in macrophages stimulated with LPS and interferon-
,10 different stimulatory conditions may have contributed to the discrepancy.
Regarding the molecular targets for ketamine-induced inhibition of TNF- and PGE2 production in glial cells, involvement of NMDA receptors in these effects is unlikely for the following reasons. First, D-AP5 failed to mimic the effects of ketamine, exhibiting only a minor inhibition of TNF-
production. Secondly, the blocking action of ketamine on NMDA receptors should be saturated at much lower concentrations than those used in our study.32 Thirdly, NMDA receptors are not considered to be expressed on most astrocytes or microglia.33 34 It is possible that NMDA receptors play minor roles in LPS-induced TNF-
production; however, these receptors do not seem to be the primary sites responsible for the observed effects of ketamine. The exact mechanism of the action of ketamine action on glial cells remains to be determined.
To examine the effects of propofol, we chose 30 µM as the clinical concentration and 300 µM as the pharmacological concentration, because the 95% effective concentration of propofol for loss of consciousness in patients is reportedly around 30 µM in the total fraction combining free and plasma binding fractions.35 We found that propofol had no effect on LPS-induced nitric oxide or TNF- production in mixed glial cells at either the clinically relevant concentration or a concentration 10 times greater. We did not examine the effects of propofol on each cell type, because the results in mixed glial cells were negative. Our results indicate that TNF-
release from primary cultures of glial cells is differentially modulated by ketamine and propofol. Whereas propofol at a concentration of 157 µM reportedly increased LPS-stimulated TNF-
production in human whole blood,12 it was shown to inhibit nitric oxide and TNF-
release from alveolar macrophages in an endotoxin-induced lung injury model14 and to suppress nitric oxide and iNOS expression in LPS-stimulated macrophages at 25100 µM.36 A number of factors could account for the different results obtained in different experiments including cell-specific differences in the regulation of inflammatory responses mentioned above, differences in culture conditions (i.e. whole-blood culture containing heparin vs the usual culture system), and differences in in vivo and in vitro experiments. Further study is required to clarify the reasons for the difference in propofol action in different cells.
Quantification of nitrite by the Griess reaction has some limitations. It measures nitrite, the major product of nitric oxide, but not nitrate. It is possible that the nitrite concentrations may be a fraction of total nitric oxide released. Another method, such as converting nitrate to nitrite or measuring iNOS expression, could be used to circumvent this problem, but the Griess reaction has been widely used to monitor nitric oxide production in biological fluids. Because other studies reporting inhibitory effects of ketamine and propofol on nitric oxide release from immune cells also used the Griess reaction to estimate nitric oxide release,10 36 the different findings between earlier and current reports cannot be explained by limitations of this method.
This study simulates infection of the CNS with gram-negative bacteria, and our results suggest that ketamine may attenuate some of the inflammatory responses of glial cells in this pathological condition. Inflammatory responses of glial cells also develop in various forms of brain injury, including stroke and trauma. Spinal glial inflammation is also believed to play a role in exaggerated pain states.5 Glial inflammatory responses are relatively stereotypic1 and relevant to patients suffering from these various insults to the CNS. In these situations, the proinflammatory cytokines released, such as TNF- and interleukin 1ß, also activate glial cells via toll-like receptors and downstream signalling pathways partially shared by LPS-stimulated signalling.2 This study raised the possibility that ketamine might modulate some of the inflammatory processes in these pathological conditions. However, further studies are needed to clarify whether ketamine attenuates the response of glial cells to proinflammatory cytokines and whether it exerts anti-inflammatory effects on glial cells in vivo.
In conclusion, we found that ketamine inhibited some of the inflammatory responses of both astrocytes and microglial cells treated with LPS without causing major changes in nitric oxide release. In contrast, propofol did not affect LPS-induced TNF- or nitric oxide release from glial cells.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 Nguyen MD, Julien JP, Rivest S. Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat Rev Neurosci 2002; 3: 21627[CrossRef][ISI][Medline]
3 Gonzalez-Scarano F, Baltuch G. Microglia as mediators of inflammatory and degenerative diseases. Annu Rev Neurosci 1999; 22: 21940[CrossRef][ISI][Medline]
4 Barone FC, Feuerstein GZ. Inflammatory mediators and stroke: new opportunities for novel therapeutics. J Cereb Blood Flow Metab 1999; 19: 81934[CrossRef][ISI][Medline]
5 Watkins LR, Maier SF. Glia: a novel drug discovery target for clinical pain. Nat Rev Drug Discov 2003; 2: 97385[CrossRef][ISI][Medline]
6 Loihl AK, Murphy S. Expression of nitric oxide synthase-2 in glia associated with CNS pathology. Prog Brain Res 1998; 118: 25367[ISI][Medline]
7 Tikka T, Fiebich BL, Goldsteins G, Keinanen R, Koistinaho J. Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci 2001; 21: 25808
8 Wu DC, Jackson-Lewis V, Vila M et al. Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci 2002; 22: 176371
9 Takenaka I, Ogata M, Koga K, Matsumoto T, Shigematsu A. Ketamine suppresses endotoxin-induced tumor necrosis factor alpha production in mice. Anesthesiology 1994; 80: 4028[ISI][Medline]
10 Shimaoka M, Iida T, Ohara A et al. Ketamine inhibits nitric oxide production in mouse-activated macrophage-like cells. Br J Anaesth 1996; 77: 23842
11 Kawasaki T, Ogata M, Kawasaki C et al. Ketamine suppresses proinflammatory cytokine production in human whole blood in vitro. Anesth Analg 1999; 89: 6659
12 Larsen B, Hoff G, Wilhelm W et al. Effect of intravenous anesthetics on spontaneous and endotoxin-stimulated cytokine response in cultured human whole blood. Anesthesiology 1998; 89: 121827[CrossRef][ISI][Medline]
13 Taniguchi T, Yamamoto K, Ohmoto N, Ohta K, Kobayashi T. Effects of propofol on hemodynamic and inflammatory responses to endotoxemia in rats. Crit Care Med 2000; 28: 11016[CrossRef][ISI][Medline]
14 Gao J, Zeng BX, Zhou LJ, Yuan SY. Protective effects of early treatment with propofol on endotoxin-induced acute lung injury in rats. Br J Anaesth 2004; 92: 2779
15 Pahan K, Namboodiri AM, Sheikh FG, Smith BT, Singh I. Increasing cAMP attenuates induction of inducible nitric-oxide synthase in rat primary astrocytes. J Biol Chem 1997; 272: 778691
16 Pahan K, Sheikh FG, Namboodiri AM, Singh I. Inhibitors of protein phosphatase 1 and 2A differentially regulate the expression of inducible nitric-oxide synthase in rat astrocytes and macrophages. J Biol Chem 1998; 273: 1221926
17 Watters JJ, Sommer JA, Pfeiffer ZA et al. A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production. J Biol Chem 2002; 277: 907787
18 O'Banion MK. Cyclooxygenase-2: molecular biology, pharmacology, and neurobiology. Crit Rev Neurobiol 1999; 13: 4582[ISI][Medline]
19 Vanegas H, Schaible HG. Prostaglandins and cyclooxygenases [correction of cycloxygenases] in the spinal cord. Prog Neurobiol 2001; 64: 32763[CrossRef][ISI][Medline]
20 Bezzi P, Domercq M, Brambilla L et al. CXCR4-activated astrocyte glutamate release via TNFalpha: amplification by microglia triggers neurotoxicity. Nat Neurosci 2001; 4: 70210[CrossRef][ISI][Medline]
21 Nakajima K, Shimojo M, Hamanoue M et al. Identification of elastase as a secretory protease from cultured rat microglia. J Neurochem 1992; 58: 14018[ISI][Medline]
22 Sasaki Y, Takimoto M, Oda K et al. Endothelin evokes efflux of glutamate in cultures of rat astrocytes. J Neurochem 1997; 68: 2194200[ISI][Medline]
23 Pahan K, Sheikh FG, Namboodiri AM, Singh I. Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages. J Clin Invest 1997; 100: 26719
24 Bhat NR, Zhang P, Lee JC, Hogan EL. Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures. J Neurosci 1998; 18: 163341
25 Hamba M, Onodera K, Takahashi T. Long-term potentiation of primary afferent neurotransmission at trigeminal synapses of juvenile rats. Eur J Neurosci 2000; 12: 112834[CrossRef][ISI][Medline]
26 Green LC, Wagner DA, Glogowski J et al. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982; 126: 1318[CrossRef][ISI][Medline]
27 Balazs R, Jorgensen OS, Hack N. N-methyl-D-aspartate promotes the survival of cerebellar granule cells in culture. Neuroscience 1988; 27: 43751[CrossRef][ISI][Medline]
28 Idvall J, Ahlgren I, Aronsen KR, Stenberg P. Ketamine infusions: pharmacokinetics and clinical effects. Br J Anaesth 1979; 51: 116773[ISI][Medline]
29 Cohen ML, Chan SL, Way WL, Trevor AJ. Distribution in the brain and metabolism of ketamine in the rat after intravenous administration. Anesthesiology 1973; 39: 3706[ISI][Medline]
30 Domino EF, Zsigmond EK, Domino LE et al. Plasma levels of ketamine and two of its metabolites in surgical patients using a gas chromatographic mass fragmentographic assay. Anesth Analg 1982; 61: 8792[Abstract]
31 Li CY, Chou TC, Wong CS et al. Ketamine inhibits nitric oxide synthase in lipopolysaccharide-treated rat alveolar macrophages. Can J Anaesth 1997; 44: 98995[Abstract]
32 Zeilhofer HU, Swandulla D, Geisslinger G, Brune K. Differential effects of ketamine enantiomers on NMDA receptor currents in cultured neurons. Eur J Pharmacol 1992; 213: 1558[CrossRef][ISI][Medline]
33 Porter JT, McCarthy KD. Astrocytic neurotransmitter receptors in situ and in vivo. Prog Neurobiol 1997; 51: 43955[CrossRef][ISI][Medline]
34 Noda M, Nakanishi H, Nabekura J, Akaike N. AMPA-kainate subtypes of glutamate receptor in rat cerebral microglia. J Neurosci 2000; 20: 2518
35 Smith C, McEwan AI, Jhaveri R et al. The interaction of fentanyl on the Cp50 of propofol for loss of consciousness and skin incision. Anesthesiology 1994; 81: 8208[ISI][Medline]
36 Chen RM, Wu GJ, Tai YT et al. Propofol reduces nitric oxide biosynthesis in lipopolysaccharide-activated macrophages by downregulating the expression of inducible nitric oxide synthase. Arch Toxicol 2003; 77: 41823[CrossRef][ISI][Medline]