Soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid is neuroprotective in rat model of ischemic stroke

Abstract

Soluble epoxide hydrolase (sEH) diminishes vasodilatory and neuroprotective effects of epoxyeicosatrienoic acids by hydrolyzing them to inactive dihydroxy metabolites. The primary goals of this study were to investigate the effects of acute sEH inhibition by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on infarct volume, functional outcome, and changes in cerebral blood flow (CBF) in a rat model of ischemic stroke. Focal cerebral ischemia was induced in rats for 90 min followed by reperfusion. At the end of 24 h after reperfusion rats were euthanized for infarct volume assessment by triphenyltetrazolium chloride staining. Brain cortical sEH activity was assessed by ultra performance liquid chromatography-tandem mass spectrometry. Functional outcome at 24 and 48 h after reperfusion was evaluated by arm flexion and sticky-tape tests. Changes in CBF were assessed by arterial spin-labeled-MRI at baseline, during ischemia, and at 180 min after reperfusion. Neuroprotective effects of t-AUCB were evaluated in primary rat neuronal cultures by Cytotox-Flour kit and propidium iodide staining. t-AUCB significantly reduced cortical infarct volume by 35% (14.5 ± 2.7% vs. 41.5 ± 4.5%), elevated cumulative epoxyeicosatrienoic acids-to-dihydroxyeicosatrienoic acids ratio in brain cortex by twofold (4.40 ± 1.89 vs. 1.97 ± 0.85), and improved functional outcome in arm-flexion test (day 1: 3.28 ± 0.5 s vs. 7.50 ± 0.9 s; day 2: 1.71 ± 0.4 s vs. 5.28 ± 0.5 s) when compared with that of the vehicle-treated group. t-AUCB significantly reduced neuronal cell death in a dose-dependent manner (vehicle: 70.9 ± 7.1% vs. t-AUCB0.1μM: 58 ± 5.11% vs. t-AUCB0.5μM: 39.9 ± 5.8%). These findings suggest that t-AUCB may exert its neuroprotective effects by affecting multiple components of neurovascular unit including neurons, astrocytes, and microvascular flow.

Keywords: arachidonic acid; cerebral blood flow; cytochrome P-450; ischemic stroke; middle cerebral artery occlusion.

Fig. 1.

The effect of acute trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) treatment on brain infarct volume after temporary middle cerebral artery occlusion (MCAO) in rats (n = 9). A: representative rat brain sections stained with 2,3,5-triphenyltetrazolium chloride following vehicle (HPβCD) or t-AUCB. B: percent infarct volume in rats treated with vehicle (HPβCD) or t-AUCB. Percent infarct volume was calculated by dividing infarct volume by contralateral hemisphere volume. Rats were euthanized 24 h after MCAO, and brain sections were obtained for infarct volume determination. Data represented as means ± SD. ***Significant values for P < 0.01.

Fig. 2.

The effect of acute t-AUCB treatment on brain cortical soluble epoxide hydrolase (sEH) activity after temporary MCAO in rats (n = 6). Rats treated with t-AUCB showed a significant increase in the ratio of cumulative epoxyeicosatrienoic acids (EETs)/dihydroxyeicosatrienoic acids (DHETs) (11,12- and 14,15- EET/DHET) (A) but no significant changes in 20-hydroxyeicosatetraenoic acids (HETE; B), 6-Keto-PGF_1α (metabolite of PGI₂; C), and PGF_2α (D) when compared with the vehicle-treated group. Brain cortices were collected at 6 h after t-AUCB dosing. Data represented as means ± SD. *Significant values for P < 0.05.

Fig. 3.

Representative LC/MS chromatograms of rat brain cortical sample showing EETs and 20-HETE before (A) and (B) treatment with t-AUCB.

Fig. 4.

The effect of acute t-AUCB on functional outcome in a rat temporary MCAO model (n = 9). Rats treated with t-AUCB showed significant improvement in arm flexure (A), neurological deficit score (B), time to remove tape (C), and tape surface area ratio (D) compared with vehicle-treated group on both days 1 and 2. Data represented as means ± SD. *Significant values for P < 0.05; ***significant values for for P < 0.001.

Fig. 5.

Comparison of cerebral blood flow (CBF) in the cerebral cortex ipsilateral to infarct of vehicle and t-AUCB-treated rats (n = 7). A: representative brain CBF maps of rats treated with vehicle or t-AUCB before MCAO (pre), during MCAO (70 min), and after post-ischemic reperfusion (270 min). Dark blue area on right cerebral cortex signifies formation of infarct. B: CBF values (reported as ml/100 g tissue/min) in the cortex ipsilateral to infarct. C: physiological parameters [mean arterial blood pressure (MABP) and blood pCO₂]. Data represented as means ± SD.

Fig. 6.

The effect of t-AUCB on cytotoxicity of primary rat cortical neuronal cultures. Cytotoxicity was assessed by using a Cytotox-Flour kit and propidium iodide (PI) staining. Cytotoxicity was represented as percent cell death normalized to positive control staurosporine (SP). A: percent cell death with t-AUCB or vehicle treatment under hypoxic and nonhypoxic conditions. B: representative images of rat primary cortical neuronal cultures imaged under fluorescence microscope after Hoechst (blue) and PI (red) staining. C: cytotoxicity assessment after hypoxic injury by PI staining. Staurosporine and MK-801 were used as positive and negative controls. Data represented as means ± SD. **Significant values for P < 0.01; ***significant values for P < 0.001, respectively.