# Inhibitory Effects of Ketamine on Lipopolysaccharide-Induced Microglial Activation

Authors: Yi Chang; Jie-Jen Lee; Cheng-Ying Hsieh; George Hsiao; Duen-Suey Chou; Joen-Rong Sheu
Journal: Mediators of Inflammation
 (2009)

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## Abstract

Microglia activated in response to brain injury release neurotoxic factors including nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor-α
(TNF-α) and interleukin-1β (IL-1β). Ketamine, an anesthetic induction agent, is generally reserved for use in patients with severe hypotension or respiratory depression. In this study, we found that ketamine (100 and 250 μM) concentration-dependently inhibited lipopolysaccharide (LPS)-induced NO and IL-1β release in primary cultured microglia. However, ketamine (100 and 250 μM) did not significantly inhibit the LPS-induced TNF-α production in microglia, except at the higher concentration (500 μM). Further study of the molecular mechanisms revealed that ketamine markedly inhibited extracellular signal-regulated kinase (ERK1/2) phosphorylation but not c-Jun N-terminal kinase or p38 mitogen-activated protein kinase stimulated by LPS in microglia. These results suggest that microglial inactivation by ketamine is at least partially due to inhibition of ERK1/2 phosphorylation.


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## Body

## 1. Introduction

Microglia are a  type of neuroglia that support, nurture, and
protect neurons which maintain homeostasis of the fluid that bathes neurons [1]. It is an innate immune component of the central
nervous system (CNS) parenchyma [2]. Under  physiological conditions, residential
microglia are quiescent and scattered throughout the CNS. Occasionally,
microglia are moderately activated to play the classic role as scavengers for
maintaining and restoring the CNS. Activated microglia release proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)  to induce inflammatory
responses [3].
Furthermore, they also release neurotoxins like reactive
oxygen species (ROS) and nitric oxide (NO) [4], which amplify the
inflammatory responses and cause neuronal damage in the CNS. Sustained overactivation of
microglia is found in many neurodegenerative diseases such as multiple
sclerosis, Alzheimer’s disease, Parkinson’s disease, HIV-associated dementia,
and ischemia/reperfusion brain injury [5, 6].Endotoxins are high-molecular-weight
complexes of lipopolysaccharide (LPS) that are major components of the outer
membranes of the cell walls of gram-negative bacteria [7]. The most severe
septic microvascular inflammatory responses, however, are caused by
gram-negative bacteriemia, and these responses can be produced by an injection
of LPS. Endothelial injury, activation of the coagulation cascade, platelet
aggregation, and thrombocytopenia have all been shown to contribute to vascular
fibrin deposition in LPS-induced septic shock
[7–9]. LPS also triggers a series of
inflammatory reactions in microglia. LPS is known to induce NO and TNF-α  production in
microglia through various extracellular signal-regulated kinase (ERK), p38
mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK)
pathways [10, 11].Ketamine, an anesthetic induction
agent, is generally reserved for use in patients with severe hypotension or
respiratory depression [12]. As we described previously, ketamine (200 and 350 μM)
significantly inhibited platelet activation stimulated by collagen [13]. On the
other hand, ketamine has been reported to exert anti-inflammatory effects on
macrophages and leucocytes in in vitro
and in vivo studies
[14–16]. Shibakawa et al. [17] also reported that ketamine
(300 ~ 1000 μM)
significantly inhibited some of the inflammatory responses in microglial cells
stimulated by LPS. However,
the detailed mechanisms underlying the anti-inflammatory effects of ketamine in
microglia stimulated by LPS still
have not been completely resolved yet. We therefore further examined the effect of ketamine in LPS-induced
microglial activation in primary cultures from rats and utilized the
findings to further characterize the anti-inflammatory effects of ketamine.


## 2. Materials and Methods

### 2.1. Materials

Ketamine, LPS (Escherichia
coli, serotype 0127: B8), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) were purchased from Sigma (St. Louis, Mo, USA); RPMI-1640 medium,
fetal bovine serum (FBS), trypsin (0.25%), L-glutamine, and penicillin/streptomycin
were from GibcoBRL (Gaithersburg, Md, USA); deoxyribonuclease
type I (DNase I) was from Roche (Indianapolis, Ind, USA); anti-ERK1/2, anti-phospho-ERK1/2
(Thr202/Tyr204), anti-JNK1/2, anti-p38 MAPK, and anti-phospho-JNK1/2 (Thr183/Tyr185)
monoclonal antibodies (mAbs) were from Cell Signaling (Beverly, Mass,
USA); the anti-phospho-p38 MAPK (Ser182)
mAb was from Santa Cruz (Santa Cruz, Calif, USA); the horseradish
peroxidase-conjugated secondary antibody was from Amersham (Buckinghamshire, UK); and the IL-1β and TNF-α enzyme immunoassay (EIA) kits were from
R&D Systems (Minneapolis, Minn, USA).


### 2.2. Cell Cultivation

Wistar rats (7
days old; from the Experimental Animal Center, College of Medic ine, National Taiwan
University) were used in this study. All animal experiments and care were
performed according to theGuide for the
Care and Use of Laboratory Animals (National Academy Press, Washington, DC,
1996). Wistar rats were
deeply anesthetized with ether and transcardially perfused with normal saline
until the lungs and liver were clear of blood. After perfusion, the brain was
removed and kept in RPMI-1640 medium. After dissecting the meninges, the brain
tissue was minced in ice-cold RPMI-1640 and treated with trypsin (0.25%) and
deoxyribonuclease (10 mg mL−1) in RPMI-1640 for 2 hours at 37°C. Treated
tissues were further minced in 10% FBS and centrifuged at 1000 rpm for 10 minutes.
The tissue pellet was resuspended in RPMI-1640 and then seeded in 75 cm2 flasks
at 37°C (95%  O2  and 5% CO2).Microglia were harvested from flasks of
mixed glial cultures after shaking for 2 hours. Cells were collected by
centrifugation and then seeded at5×105 cells mL−1.
After incubation for 1 hour at 37°C, nonadherent or weakly adherent cells were
removed by gentle shaking and washed out. Cells were further cultured in
RPMI-1640 supplemented with 10% FBS for 1 day. Approximately 2×106 cells were obtained per brain used [18]. To determine the purity of the
microglia, an immunocytochemical analysis was carried out using a microglial-specific
OX-42 antibody. These primary cultured cells were >95% OX-42-positive indicating that they were
composed of microglia.


### 2.3. Cell Viability

Microglial viability after 24 hours
of continuous exposure to different concentrations of LPS (1 ~ 200 ng mL−1) and ketamine (100 ~ 500 μM) was measured with a colorimetric assay
based on the ability of mitochondria in viable cells to reduce the MTT as previously
described [19]. The percentage of cell viability was calculated as the
absorbance of treated cells/control cells × 100%.


### 2.4. Determination of Nitrite Concentration

To determine NO production from
microglia, nitrite (a stable oxidative end product of NO) accumulation in the
media of microglia was measured using a colorimetric method [18], with minor modifications. Briefly, 100 μL of supernatant was incubated with an equal
volume of Griess reagent (1% sulfanilamide and 0.1% naphthyl-ethylenediamine
dihydrocholoride in 2.5% phosphoric acid). After a 30-minute incubation at room
temperature, the optical absorbance at 550 nm was measured with a microplate
reader. Nitrite concentrations were calculated by regression with standard
solutions of sodium nitrite prepared in the same culture medium
[18].


### 2.5. Enzyme-Linked Immunosorbent Assay (ELISA) of IL-1β and TNF-α

For determination of IL-1β and TNF-α production from microglia, microglia
(5×105 cells mL−1) were plated onto 24-well culture plates for 24 hours. Cells were
pretreated with various
concentrations of ketamine (100 ~ 500 μM) or an isovolumetric PBS buffer for 30 minutes and then
treated with LPS (100 ng mL−1) for the indicated times (1 ~ 24 hours). After incubation with LPS,
supernatants were collected and immediately frozen at −70°C. The IL-1β and TNF-α levels of the supernatants were measured using ELISA kits according to the
manufacturer’s protocol.


### 2.6. Western Blot Analysis

For determination of the
expression of MAPKs in microglia, Western blot analyses were performed as
described previously [18]. Microglia (5×105 cells mL−1) were cultured on 24-well plates and treated
with ketamine
(100 and 250 μM) or an isovolumetric
PBS buffer for 30 minutes followed by the addition of LPS (100 ng mL−1). At indicated times, cells were washed with
ice-cold PBS buffer (pH 7.3). Proteins were extracted with lysis buffer for 30
minutes. In addition, phosphatase inhibitors (10 mM sodium fluoride, 1 mM sodium orthovanadate, and 10 mM sodium pyrophosphate) were added to the lysis
buffer for the phosphorylated MAPK analysis. Lysates were centrifuged, and the
supernatant (50 μg protein) was subjected to SDS-PAGE and electrophoretically
transferred onto PVDF membranes (0.45 mm;
Hybond-P; Amersham). After incubation in blocking buffer
(50 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20, and 5% dry skim milk; pH 7.5) overnight
at 48 hours and being washed three times with
PBS buffer, blots were treated with either anti-phospho-ERK1/2 (p42/44),
anti-ERK1/2, anti-phospho-JNK1/2 (p46/54), anti-JNK1/2, anti-p38 MAPK, or anti-phospho-p38 MAPK mAbs (1:2000) in a PBS
buffer for 3 hours. They were subsequently washed three times with PBS buffer
and incubated with a peroxidase-conjugated goat anti-mouse or anti-rabbit antibody
(1:3000) for 2 hours. Blots were then washed three times, and the band with
peroxidase activity was detected using film exposure with enhanced
chemiluminescence detection reagents (ECL+ system; Amersham). Densitometric analysis
of specific bands was performed using a Photo-Print Digital Imaging System
(IP-008-SD) with analytic software (Bio-1Dlight, Vers. 2000).


### 2.7. Statistical Analysis

The experimental results are expressed as the means ± S.E.M. and are accompanied by the number of observations.
Data were assessed by the
method of analysis of variance (ANOVA). If a significant difference among the
group means was noted, the difference between two groups would be assessed using
the Newman-Keuls method. AP value of <.05 was considered statistically significant.


## 2.1. Materials

Ketamine, LPS (Escherichia
coli, serotype 0127: B8), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) were purchased from Sigma (St. Louis, Mo, USA); RPMI-1640 medium,
fetal bovine serum (FBS), trypsin (0.25%), L-glutamine, and penicillin/streptomycin
were from GibcoBRL (Gaithersburg, Md, USA); deoxyribonuclease
type I (DNase I) was from Roche (Indianapolis, Ind, USA); anti-ERK1/2, anti-phospho-ERK1/2
(Thr202/Tyr204), anti-JNK1/2, anti-p38 MAPK, and anti-phospho-JNK1/2 (Thr183/Tyr185)
monoclonal antibodies (mAbs) were from Cell Signaling (Beverly, Mass,
USA); the anti-phospho-p38 MAPK (Ser182)
mAb was from Santa Cruz (Santa Cruz, Calif, USA); the horseradish
peroxidase-conjugated secondary antibody was from Amersham (Buckinghamshire, UK); and the IL-1β and TNF-α enzyme immunoassay (EIA) kits were from
R&D Systems (Minneapolis, Minn, USA).


## 2.2. Cell Cultivation

Wistar rats (7
days old; from the Experimental Animal Center, College of Medic ine, National Taiwan
University) were used in this study. All animal experiments and care were
performed according to theGuide for the
Care and Use of Laboratory Animals (National Academy Press, Washington, DC,
1996). Wistar rats were
deeply anesthetized with ether and transcardially perfused with normal saline
until the lungs and liver were clear of blood. After perfusion, the brain was
removed and kept in RPMI-1640 medium. After dissecting the meninges, the brain
tissue was minced in ice-cold RPMI-1640 and treated with trypsin (0.25%) and
deoxyribonuclease (10 mg mL−1) in RPMI-1640 for 2 hours at 37°C. Treated
tissues were further minced in 10% FBS and centrifuged at 1000 rpm for 10 minutes.
The tissue pellet was resuspended in RPMI-1640 and then seeded in 75 cm2 flasks
at 37°C (95%  O2  and 5% CO2).Microglia were harvested from flasks of
mixed glial cultures after shaking for 2 hours. Cells were collected by
centrifugation and then seeded at5×105 cells mL−1.
After incubation for 1 hour at 37°C, nonadherent or weakly adherent cells were
removed by gentle shaking and washed out. Cells were further cultured in
RPMI-1640 supplemented with 10% FBS for 1 day. Approximately 2×106 cells were obtained per brain used [18]. To determine the purity of the
microglia, an immunocytochemical analysis was carried out using a microglial-specific
OX-42 antibody. These primary cultured cells were >95% OX-42-positive indicating that they were
composed of microglia.


## 2.3. Cell Viability

Microglial viability after 24 hours
of continuous exposure to different concentrations of LPS (1 ~ 200 ng mL−1) and ketamine (100 ~ 500 μM) was measured with a colorimetric assay
based on the ability of mitochondria in viable cells to reduce the MTT as previously
described [19]. The percentage of cell viability was calculated as the
absorbance of treated cells/control cells × 100%.


## 2.4. Determination of Nitrite Concentration

To determine NO production from
microglia, nitrite (a stable oxidative end product of NO) accumulation in the
media of microglia was measured using a colorimetric method [18], with minor modifications. Briefly, 100 μL of supernatant was incubated with an equal
volume of Griess reagent (1% sulfanilamide and 0.1% naphthyl-ethylenediamine
dihydrocholoride in 2.5% phosphoric acid). After a 30-minute incubation at room
temperature, the optical absorbance at 550 nm was measured with a microplate
reader. Nitrite concentrations were calculated by regression with standard
solutions of sodium nitrite prepared in the same culture medium
[18].


## 2.5. Enzyme-Linked Immunosorbent Assay (ELISA) of IL-1β and TNF-α

For determination of IL-1β and TNF-α production from microglia, microglia
(5×105 cells mL−1) were plated onto 24-well culture plates for 24 hours. Cells were
pretreated with various
concentrations of ketamine (100 ~ 500 μM) or an isovolumetric PBS buffer for 30 minutes and then
treated with LPS (100 ng mL−1) for the indicated times (1 ~ 24 hours). After incubation with LPS,
supernatants were collected and immediately frozen at −70°C. The IL-1β and TNF-α levels of the supernatants were measured using ELISA kits according to the
manufacturer’s protocol.


## 2.6. Western Blot Analysis

For determination of the
expression of MAPKs in microglia, Western blot analyses were performed as
described previously [18]. Microglia (5×105 cells mL−1) were cultured on 24-well plates and treated
with ketamine
(100 and 250 μM) or an isovolumetric
PBS buffer for 30 minutes followed by the addition of LPS (100 ng mL−1). At indicated times, cells were washed with
ice-cold PBS buffer (pH 7.3). Proteins were extracted with lysis buffer for 30
minutes. In addition, phosphatase inhibitors (10 mM sodium fluoride, 1 mM sodium orthovanadate, and 10 mM sodium pyrophosphate) were added to the lysis
buffer for the phosphorylated MAPK analysis. Lysates were centrifuged, and the
supernatant (50 μg protein) was subjected to SDS-PAGE and electrophoretically
transferred onto PVDF membranes (0.45 mm;
Hybond-P; Amersham). After incubation in blocking buffer
(50 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20, and 5% dry skim milk; pH 7.5) overnight
at 48 hours and being washed three times with
PBS buffer, blots were treated with either anti-phospho-ERK1/2 (p42/44),
anti-ERK1/2, anti-phospho-JNK1/2 (p46/54), anti-JNK1/2, anti-p38 MAPK, or anti-phospho-p38 MAPK mAbs (1:2000) in a PBS
buffer for 3 hours. They were subsequently washed three times with PBS buffer
and incubated with a peroxidase-conjugated goat anti-mouse or anti-rabbit antibody
(1:3000) for 2 hours. Blots were then washed three times, and the band with
peroxidase activity was detected using film exposure with enhanced
chemiluminescence detection reagents (ECL+ system; Amersham). Densitometric analysis
of specific bands was performed using a Photo-Print Digital Imaging System
(IP-008-SD) with analytic software (Bio-1Dlight, Vers. 2000).


## 2.7. Statistical Analysis

The experimental results are expressed as the means ± S.E.M. and are accompanied by the number of observations.
Data were assessed by the
method of analysis of variance (ANOVA). If a significant difference among the
group means was noted, the difference between two groups would be assessed using
the Newman-Keuls method. AP value of <.05 was considered statistically significant.


## 3. Results

According to a preliminary
test, activation of microglia by LPS (100 ng mL−1) induced a significant
and marked increase in nitrite formation. Therefore, an LPS concentration of 100 ng mL−1 was employed in the following experiments. In
this study, the concentration of nitrite produced in the cell supernatant
time-dependently increased from 0.5±0.0 (resting) to 6.7±0.3 μM at 24 hours after LPS treatment (Figure 1).  Ketamine (100 and 250 μM) concentration-dependently inhibited LPS-(100 ng mL−1) stimulated nitrite production by
approximately 40% and 60%, respectively, (Figure 1). Ketamine neither interfered
with the Griess reaction nor reacted with native NO (data not shown). These
results demonstrate that ketamine markedly suppressed NO production stimulated
by LPS in microglia. Furthermore, neither ketamine (100 ~ 500 μM) (Figure 2) nor LPS (100 ng mL−1) (data not shown) significantly affected
the cell viability of microglia for 24 hours according to the MTT assay.Figure 1
Effect of ketamine on nitrite formation
in LPS-activated microglia. Microglia (5×105 cells mL−1)
were treated with ketamine (100 and 250 μM) or an isovolumetric PBS buffer for 30 minutes, followed by
the addition of LPS (100 ng mL−1) for 24 hours. Cell-free
supernatants were assayed for nitrite production as described
in Section 2. Data are presented as the means ± S.E.M. (n=3). P<.01**, compared to the
resting group; P<.01##, compared to
the PBS-treated group.Figure 2
Tetrazolium dye 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay of ketamine-treated microglia. Microglia (5×105 cells mL−1)  were
treated with various concentrations of ketamine (100 ~ 500 μM) or an isovolumetric PBS buffer for 24 hours.
Cell viability was measured by a colorimetric assay at 550 nm based on the
ability of mitochondria to reduce the MTT in viable cells. Data are
presented as the means ± S.E.M. (n=3).LPS (100 ng mL−1) induced a
time-dependent increase in IL-1β formation in microglia,
and it reached a maximal level at 12 hours rising from 135.9±6.5 (resting) to 575.9±10.6 pg mL−1 (Figure 3). On
the other hand, after pretreatment of cells with various
concentrations of ketamine for
30  minutes followed by the addition of LPS (100 ng mL−1) for 12 hours, we found
that ketamine (100 and 250 μM) concentration-dependently inhibited IL-1β production by approximately
13% and 36%, respectively, (Figure 3).Figure 3
Effect of ketamine
on interleukin (IL)-1β production in lipopolysaccharide (LPS)-activated microglia. Microglia
(5×105 cells mL−1) were treated with ketamine (100 and 250 μM) or an isovolumetric PBS buffer
for 30 minutes, followed by the addition of LPS
(100 ng mL−1) for 12
hours. Cell-free supernatants were assayed for IL-1β production as described in Section 2. Data are
presented as the means ± S.E.M. (n=3). P<.01**, compared to the
resting group; P#<.05  and P##<.01, compared to
the PBS-treated group.On the other hand, activation
of microglia by LPS (100 ng mL−1) induced a significant increase in TNF-α formation. The peak activation of TNF-α formation occurred 3 hours after LPS (100 ng mL−1)  stimulation (resting, 1042.2±8.8 pg mL−1; 3
hours, 1992.8±38.2 pg mL−1, n=3) (Figure 4). After pretreating cells with
various concentrations of ketamine for 30 minutes followed by the addition of
LPS (100 ng mL−1)
for 3 hours, only ketamine at the higher concentration (500 μM) significantly
inhibited TNF-α production by approximately
11% (Figure 4).Figure 4
Effect of ketamine on tumor necrosis
factor (TNF)-α production in lipopolysaccharide (LPS)-activated
microglia. Microglia (5×105 cells mL−1) were treated
with various concentrations of ketamine (100, 250, and 500 μM) or an isovolumetric
PBS buffer for 30 minutes, followed
by the addition of LPS (100 ng mL−1) for 3 hours. Cell-free
supernatants were assayed for TNF-α production as described in Section 2. Data are presented as the means ± S.E.M. (n=3). P<.01**, compared to the
resting group; P#<.05, compared to
the PBS-treated group.To further investigate the inhibitory
mechanisms of ketamine in LPS-induced microglial activation, three major MAPK
signaling molecules were detected: ERK1/2, JNK1/2, and p38 MAPK. The
immunoblot analysis revealed that treatment with LPS (100 ng mL−1) induced
rapid and marked time-dependent phosphorylation of ERK1/2 (p42/p44), JNK1/2 (p46/p54),
and p38 MAPK, which reached maximal levels at approximately 45 minutes and then
returned to the basal level (data not shown). After being pretreated with ketamine
(100 and 250 μM) for 30 minutes,
phosphorylated ERK1/2 stimulated by LPS (100 ng mL−1) was
markedly inhibited by ketamine in a concentration-dependent
manner (Figure 5). However, neither JNK1/2 (Figure 6) nor p38 MAPK (Figure 7)
phosphorylation stimulated by LPS was significantly inhibited by ketamine
(100 and 250 μM).Figure 5
Effect of ketamine on ERK1/2
phosphorylation in lipopolysaccharide (LPS)-activated microglia. Microglia (5×105 cells mL−1) were treated with ketamine (100 and 250 μM) or an isovolumetric PBS buffer for
30 minutes, followed by the addition of LPS
(100 ng mL−1) for 45 minutes. ERK1/2 phosphorylation was
determined by Western blotting with a monoclonal antibody which recognizes only
phosphorylated ERK1/2 (p42/p44). Data are presented as the means ± S.E.M.
(n=3). P<.01**, compared to the
resting group; P#<.05, compared to
the PBS-treated group.Figure 6
Effect of ketamine on JNK1/2
phosphorylation in lipopolysaccharide (LPS)-activated microglia. Microglia (5×105 cells mL−1) were treated with ketamine (100 and 250 μM) or an isovolumetric PBS buffer for
30 minutes, followed by the addition of LPS
(100 ng mL−1) for 45 minutes. JNK1/2 phosphorylation was
determined by Western blotting with a monoclonal antibody which recognizes only
phosphorylated JNK1/2 (p46/p54). Data are presented as the means ± S.E.M.
(n=3).  P<.01**, compared to the
resting group.Figure 7
Effect of ketamine on p38 MAPK
phosphorylation in lipopolysaccharide (LPS)-activated microglia. Microglia (5×105 cells mL−1) were treated with ketamine (100 and 250 μM) or an isovolumetric PBS buffer for
30 minutes, followed by the addition of LPS
(100 ng mL−1) for 45 minutes. p38 MAPK phosphorylation
was determined by Western blotting with a monoclonal antibody which recognizes
only phosphorylated p38 MAPK. Data are presented as the means ± S.E.M. (n=3). P<.05*, compared to the
resting group.


## 4. Discussion

In this study, we demonstrate an anti-inflammatory effect of ketamine
in primary cultured microglia. Ketamine exhibited more potent activity at
inhibiting both NO and IL-1β formation
than TNF-α in
LPS-stimulated microglia. This
discrepancy, however, needs to be further investigated. The high concentration
(500 μM) of ketamine caused no
significant changes in the number of viable cells estimated by the MTT
reduction assay (Figure 2). This finding excluded the possibility that the
release of the inflammatory mediators was inhibited by the cytotoxic actions of
ketamine.It is known
that microglia play a key role in mediating inflammatory processes in the CNS,
which are associated with various neurodegenerative diseases. LPS, a glycolipid
derived from the membrane surface of gram-negative bacteria (i.e., an endotoxin),
can trigger a series of inflammatory reactions in phagocytes such as microglia.
With LPS stimulation, microglia are activated to drastically change their
cellular functions, producing various types of inflammatory mediators such as NO,
TNF-α, and IL-1β. NO and TNF-α are two major inflammatory
mediators. NO is beneficial as a messenger or modulator, but in conditions such
as oxidative stress, it is potentially toxic. NO generation by
activated microglia has been shown to cause excitotoxicity through inducing
glutamate release and inhibiting neuronal respiration [20]. TNF-α and IL-1β released from activated
microglia also stimulate NO production in glial cells and may have a direct
effect on neurons through activating receptors that contain the death domains involved
in apoptosis [21].In general, total plasma levels of ketamine
are reported to be
in the range of 33 ~ 94μM
immediately after 2.0 ~ 2.2 mg kg−1 i.v. administration in humans [22].
Therefore, we considered 100 μM to be
a higher clinically relevant concentration achievable during induction of
ketamine anesthesia, assuming that protein binding is comparable in
serum-supplemented media and plasma. Our studies suggest that ketamine may
modulate some of the inflammatory responses (i.e., NO and IL-1β) of
microglia stimulated by LPS in vitro at
the high range of clinically achievable concentrations.This anti-inflammatory
property of ketamine may have advantages in clinical therapy. An adequate
inflammatory reaction results from the equilibrium between proinflammatory and
anti-inflammatory influences. Ketamine administrated in a stress situation
favors this equilibrium. This is based on clinical data published several years
ago. The survival rate in intensive care unit of patients with septic shock was
improved when they received ketamine as a sedative [23].MAPKs play important roles
in mediating cytokine (i.e., TNF-α and IL-1β) release with LPS-stimulated
microglial activation [10, 11, 24]. We thus further investigated the roles of MAPKs involved in ketamine-mediated
suppression of inflammatory responses in LPS-stimulated microglia.
MAPKs are a family of serine-threonine kinases activated by many stimuli
including growth factors and hormones in proliferative cells
[25]. MAPKs are able to regulate
a number of transcription factors, cytoplasmic proteins, and downstream
kinases. This family consists of three major subgroups. ERK1/2 are involved in
proliferation, adhesion, and cell progression [25]. p38 MAPK and JNK1/2 or stress-activated protein
kinases, which include the 46-kDa JNK1 and 54-kDa JNK2 isoforms, are involved
in death signaling processes [25]. In the present study, we found that ketamine
inhibited ERK1/2 but not JNK1/2 or p38 MAPK phosphorylation in LPS-stimulated microglia.
Zhang et al. [26] have
reported that ketamine could abolish hyperglycemia-activated ERK1/2
phosphorylation through inhibition of the N-methyl D-aspartate-mediated calcium
influx, which subsequently reduces
the hyperglycemia-exaggerated damage [26].In conclusion, although
ketamine has been shown to exert anti-inflammatory effects on a variety of
immune cells, the exact mechanisms responsible for these actions are not well
understood. Our results suggest that ketamine’s anti-inflammatory activity may
be mediated, at least in part, by inhibition of ERK1/2 phosphorylation in
primary cultured microglia. However, more detailed anti-inflammatory mechanisms
of ketamine in microglia need to be further investigated and classified.



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*Source: 705379-2009-03-30.xml*