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

CA

1

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 D

NA

bind

ing

[arb

itrar

y 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 D

NA

bin

ding

[arb

itrar

y un

its]

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

TNF

mR

NA

exp

ress

ion

0

5

10

15

20

25

+ EET + 4-PCO

DHET

prod

uctio

n (p

g/ug

pro

tein)

0

10

20

30

40

*

unstimulated

LPS stimulated

+ LPS

*

sEHisolectinDAPI

isolectinNF-kB p65

DAPI

DMSO4-PCO