korneri@ohsu.edu soluble epoxide hydrolase inhibitor reduces neuronal death and nf-kb activation...
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korneri@ohsu.edu
Soluble Epoxide Hydrolase Inhibitor Reduces Neuronal Death and NF-kB Activation after Cardiac Arrest
Department of Anesthesiology & Perioperative Medicine, OHSU, Portland, OR
Wang JM, Lasarzik I, Kosaka Y, Herson PS, Koerner IP
San Diego, California, 2010
Background
Methods
Results
[1] Koerner IP et al. Front Biosci 2008; 13:2833
Cardiac arrest and cardiopulmonary resuscitation cause significant loss of hippocampal CA1 neurons. Neuronal survival can be improved by inhibition of sEH, even when treatment is initiated after resuscitation. This coincides with reduced activation of the pro-inflammatory transcription factor NF-κB in treated mice. In vitro, sEH inhibition reduces NF-κB activation in primary microglia and decreases subsequent transcription of pro-inflammatory TNFα. This suggests that improved neuronal survival after sEH inhibition in vivo is linked to altered microglial activation. Further study is needed to fully investigate the neurotoxic pathways blocked by sEH inhibition in vivo. Better understanding of these mechanisms will provide new insights into the therapeutic potential of sEH inhibition for survivors of cardiac arrest.
Soluble epoxide hydrolase (sEH), which
inactivates the arachidonic acid metabolite
epoxyeicosatrienoic acid (EET), contributes
to brain injury and inflammatory cytokine
expression after experimental stroke [1]. We
hypothesized that sEH is similarly involved
in injury and inflammation after global
cerebral ischemia. We therefore investigated
whether inhibition of sEH reduces neuronal
death in a mouse model of cardiac arrest and
cardiopulmonary resuscitation (CA/CPR). We
also tested whether activation of microglia,
the brain resident immune cells, and
subsequent injurious inflammation is
reduced by inhibition of sEH.
Inhibition of sEH reduces death of CA1 neurons after CA/CPR in mice
adult male C57BL/6 mice (20-25g)
endotracheally intubated, isoflurane anesthesia
right internal jugular vein cannulated
CA induced with 50 ul 0.5 M KCl iv → EKG asystole
during CA, body temp. is 28 Cº, head temp. 38 Cº
CPR after 8 minutes of CA (epinephrine + chest compressions)
return of spontaneous circulation (ROSC) within 2 min
sEH inhibitor 4-phenylchalcone oxide (4-PCO) or DMSO
(vehicle) is given by ip injection (5 mg/kg) 5 minutes and 24 hrs
after ROSC
brains are harvested after 24 or 72 hours after CA/CPR after
saline or 4% PFA perfusion
cortical mixed-glia cultures from postnatal C57BL/6, grown to
confluence in DMEM with 10% fetal bovine serum (FBS)
microglia are shaken off, re-plated at 2x105 cells/ml
sEH substrate 14,15-EET (1μM) is added with or without 4-PCO
(2μM) and DHET concentration within cells measured by LC/MS
4-PCO and/or 14,15 EET is added 1 hours before stimulation
with 100 U/ml lipopolysaccharide (LPS)
nuclear protein is extracted 30 minutes after stimulation; NF-κB
p65 DNA binding is measured by ELISA (Active Motif)
TNFα mRNA expression is quantified by RT-PCR (ABI Prism)
groups are compared using ANOVA or Student’s t-test; data are
mean+/- SEM
• mRNA expression of the pro-inflammatory and neurotoxic cytokine TNF-α, which is regulated by NF-κB, is increased 24 hours after LPS.
• pretreatment with 14,15-EET and 4-PCO significantly reduces TNF-α expression after LPS stimulation.
• n=3; *=p<0.05 vs. vehicle + LPS
b
Inhibition of sEH reduces NF-κB activation in stimulated microglia
sEH is expressed in microglia and is functionally active Hippocampal Mac-2 staining is not reduced in 4-PCO-treated mice
• 8 minutes of CA/CPR causes significant loss of CA1 neurons 72 hours after the insult (upper and middle panel, H&E).
• CA1 loss is significantly reduced in animals treated with 4-PCO after CPR (lower panel).
• n=11-12; *p<0.05 vs. vehicle
b
NF-κB activation in hippocampus is reduced by 4-PCO after CA/CPR
• nuclear NF-κB p65 binding is increased in hippocampus 24 hours after CA/CPR.
• NF-κB activation is reduced in mice treated with 4-PCO after CPR.
• NF-κB co-localizes with microglial marker Mac-2.
• n=2-3
• sEH co-localizes with microglial marker isolectin.
• microglia rapidly produce DHET 30 minutes after stimulation with 14,15-EET.
• sEH inhibitor 4-PCO significantly reduces DHET production.
• n=4; *p<0.05 vs. vehicle
Inhibition of sEH reduces TNFα expression in stimulated microglia
• LPS induces NF-kB p65 nuclear translocation in stimulated microglia.
• NF-κB binding increases 30 min after LPS.
• pretreatment with 14,15-EET and 4-PCO reduces NF-kB activation.
• n=6; *p<0.05 vs. vehicle, ** p<0.05 vs. LPS
• Mac-2, a marker of activated microglia, is increased in hippocampus 72 hours after CA/CPR.
• sEH inhibition does not affect Mac-2 increase after CA/CPR.
• n=11-12
Summary and Conclusion
vehicle 4-PCO
% d
ead
neur
ons
CA1
20.0
40.0
60.0
80.0
100.0
*
Sham
DMSO
4-PCO
vehicle 4-PCO
Mac
-2 p
ositi
ve a
rea
[%]
0
5
10
15
sham vehicle 4-PCO
NF
-B
DN
A b
ind
ing
[ar
bit
rary
un
its]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6P=0.014 P=0.051
CA/CPR Mac-2NF-κB p65DAPI
+ LPS
NF
-B
DN
A b
indi
ng [a
rbitr
ary
units
]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
+ EET + EET
+ 4-PCO
vehicle
#**
*
vehicle vehicle 4-PCO/EET
rela
tive
TN
F
mR
NA
exp
ress
ion
0
5
10
15
20
25
+ EET + 4-PCO
DHET
prod
uctio
n (pg
/ug pr
otein)
0
10
20
30
40
*
unstimulated
LPS stimulated
+ LPS
*
sEHisolectinDAPI
isolectinNF-kB p65
DAPI
DMSO
4-PCO
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