Soluble epoxide hydrolase inhibition reverses cognitive dysfunction in a mouse model of metabolic syndrome by modulating inflammation.

Midlife metabolic syndrome (MetS) is associated with cognitive impairment in late life. The mechanism of delayed MetS-related cognitive dysfunction (MetSCD) is not clear, but it has been linked to systemic inflammation and chronic cerebral microangiopathy. Currently there is no treatment for late life MetSCD other than early risk factor modification. We investigated the effect of soluble epoxide hydrolase (sEH) inhibitor 4-[[trans-4-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]cyclohexyl]oxy]-benzoic acid (t-AUCB) on cognitive performance, cerebral blood flow (CBF), and central and peripheral inflammation in the high-fat diet (HFD) model of MetS in mice. At 6 weeks of age, male mice were randomly assigned to receive either HFD or standard chow (STD) for 6 months. Mice received either t-AUCB or vehicle for 4 weeks. Cognitive performance was evaluated, followed by CBF measurement using magnetic resonance imaging (MRI). At the end of the study, blood was collected for measurement of eicosanoids and inflammatory cytokines. The brains were then analyzed by immunohistochemistry for glial activation markers. The HFD caused a significant impairment in novel object recognition. Treatment with t-AUCB increased plasma levels of 14,15-EET, prevented this cognitive impairment and modified hippocampal glial activation and plasma cytokine levels, without affecting CBF in mice on HFD. In conclusion, sEH inhibition for four weeks prevents cognitive deficits in mice on chronic HFD by modulating inflammatory processes without affecting CBF.

Sex Difference in Capillary Reperfusion After Transient Middle Cerebral Artery Occlusion in Diabetic Mice.

BACKGROUND: Type 2 diabetes (DM2) exacerbates stroke injury, reduces efficacy of endovascular therapy, and worsens long-term functional outcome. Sex differences exist in stroke incidence, response to therapy, poststroke microvascular dysfunction, and functional recovery. In this study, we tested the hypotheses that poor outcome after stroke in the setting of DM2 is linked to impaired microvascular tissue reperfusion and that male and female DM2 mice exhibit different microvascular reperfusion response after transient middle cerebral artery occlusion (MCAO). METHODS: Transient MCAO was induced for 60 minutes using an intraluminal filament in young adult DM2 and nondiabetic control male and female mice. Capillary flux in deep cortical layers was assessed using optical coherence tomography-based optical microangiography (OMAG), and associated regional brain infarct size was evaluated by hematoxylin and eosin staining. RESULTS: Compared to baseline, MCAO reduced absolute capillary red blood cell flux by 84% at 24 hours post-MCAO in male DM2 (P<0.001) but not male control mice. When normalized to pre-MCAO baseline, red blood cell flux 24 hours after stroke was 64% lower in male DM2 mice than male nondiabetic controls (P<0.01). In females, MCAO decreased capillary flux by 48% at 24 hours post-MCAO compared with baseline in DM2 (P<0.05) but not in control mice. Red blood cell flux of female DM2 mice did not differ from that of nondiabetic controls either before or 24 hours after MCAO. Furthermore, normalized capillary flux 24 hours after MCAO failed to differ between female DM2 mice and nondiabetic controls. Concomitantly, male but not female DM2 mice experienced 25% larger infarct in caudate-putamen versus respective nondiabetic controls (P<0.05). CONCLUSIONS: DM2 impairs capillary perfusion and exacerbates ischemic deep brain injury in male but not female young adult mice. Premenopausal females appear to be protected against DM2-related capillary dysfunction and brain injury.

GPR39 Knockout Worsens Microcirculatory Response to Experimental Stroke in a Sex-Dependent Manner.

No current treatments target microvascular reperfusion after stroke, which can contribute to poor outcomes even after successful clot retrieval. The G protein-coupled receptor GPR39 is expressed in brain peri-capillary pericytes, and has been implicated in microvascular regulation, but its role in stroke is unknown. We tested the hypothesis that GPR39 plays a protective role after stroke, in part due to preservation of microvascular perfusion. We generated GPR39 knockout (KO) mice and tested whether GPR39 gene deletion worsens capillary blood flow and exacerbates brain injury and functional deficit after focal cerebral ischemia. Stroke was induced in male and female GPR39 KO and WT littermates by 60-min middle cerebral artery occlusion (MCAO). Microvascular perfusion was assessed via capillary red blood cell (RBC) flux in deep cortical layers in vivo using optical microangiography (OMAG). Brain injury was assessed by measuring infarct size by 2,3,5-triphenyltetrazolium chloride staining at 24 h or brain atrophy at 3 weeks after ischemia. Pole and cylinder behavior tests were conducted to assess neurological function deficit at 1 and 3 weeks post-stroke. Male but not female GPR39 KO mice exhibited larger infarcts and lower capillary RBC flux than WT controls after stroke. Male GPR39 KO mice also exhibited worse neurologic deficit at 1 week post-stroke, though functional deficit disappeared in both groups by 3 weeks. GPR39 deletion worsens brain injury, microvascular perfusion, and neurological function after experimental stroke. Results indicate that GPR39 plays a sex-dependent role in re-establishing microvascular flow and limiting ischemic brain damage after stroke.

Age-dependent cognitive impairment, hydrocephalus and leukocyte infiltration in transgenic mice with endothelial expression of human EPHX2.

Soluble epoxide hydrolase (sEH) is upregulated in microvascular endothelium of human brain with vascular cognitive impairment (VCI). Transgenic endothelial expression of human sEH in mice (Tie2hsEH) induces endothelial dysfunction (ED), a pathogenetic mechanism of VCI. We sought to determine if endothelial upregulation of sEH is sufficient to cause cognitive impairment, and if cognitive impairment due to chronic hypoperfusion induced by unilateral common carotid artery occlusion (CCAO) is exacerbated in Tie2hsEH mice. Behavioral performance was assessed by the open field, rotarod, novel object, Morris water maze and fear conditioning tests. Cerebral blood flow and brain morphology were evaluated by MRI, and inflammatory changes investigated using immunohistochemistry and flow cytometry. We demonstrate that transgenic endothelial expression of sEH is sufficient to induce cognitive impairment, associated with leukocyte infiltration, brain atrophy and accelerated, age-dependent ventriculomegaly, identifying ED and sEH upregulation as potential underlying mechanisms and therapeutic targets for VCI.

GPR39 Deficiency Impairs Memory and Alters Oxylipins and Inflammatory Cytokines Without Affecting Cerebral Blood Flow in a High-Fat Diet Mouse Model of Cognitive Impairment.

Vascular cognitive impairment (VCI) is the second most common cause of dementia. There is no treatment for VCI, in part due to a lack of understanding of the underlying mechanisms. The G-protein coupled receptor 39 (GPR39) is regulated by arachidonic acid (AA)-derived oxylipins that have been implicated in VCI. Furthermore, GPR39 is increased in microglia of post mortem human brains with VCI. Carriers of homozygous GPR39 SNPs have a higher burden of white matter hyperintensity, an MRI marker of VCI. We tested the hypothesis that GPR39 plays a protective role against high-fat diet (HFD)-induced cognitive impairment, in part mediated via oxylipins actions on cerebral blood flow (CBF) and neuroinflammation. Homozygous (KO) and heterozygous (Het) GPR39 knockout mice and wild-type (WT) littermates with and without HFD for 8 months were tested for cognitive performance using the novel object recognition (NOR) and the Morris water maze (MWM) tests, followed by CBF measurements using MRI. Brain tissue and plasma oxylipins were quantified with high-performance liquid chromatography coupled to mass spectrometry. Cytokines and chemokines were measured using a multiplex assay. KO mice, regardless of diet, swam further away from platform location in the MWM compared to WT and Het mice. In the NOR test, there were no effects of genotype or diet. Brain and plasma AA-derived oxylipins formed by 11- and 15-lipoxygenase (LOX), cyclooxygenase (COX) and non-enzymatically were increased by HFD and GPR39 deletion. Interleukin-10 (IL-10) was lower in KO mice on HFD than standard diet (STD), whereas IL-4, interferon γ-induced protein-10 (IP-10) and monocyte chemotactic protein-3 (MCP-3) were altered by diet in both WT and KO, but were not affected by genotype. Resting CBF was reduced in WT and KO mice on HFD, with no change in vasoreactivity. The deletion of GPR39 did not change CBF compared to WT mice on either STD or HFD. We conclude that GPR39 plays a role in spatial memory retention and protects against HFD-induced cognitive impairment in part by modulating inflammation and AA-derived oxylipins. The results indicate that GPR39 and oxylipin pathways play a role and may serve as therapeutic targets in VCI.

Identification and Characterization of Small-Molecule IRF3-Dependent Immune Activators for Pharmaceutical Development.

We sought to develop a small-molecule activator of interferon regulatory factor 3 (IRF3), an essential innate immune transcription factor, which could potentially be used therapeutically in multiple disease settings. Using a high-throughput screen, we identified small-molecule entities that activate a type I interferon response, with minimal off-target NFκB activation. We identified 399 compounds at a hit rate of 0.24% from singlicate primary screening. Secondary screening included the primary hits and additional compounds with similar chemical structures obtained from other library sources and resulted in 142 candidate compounds. The hit compounds were sorted and ranked to identify compound groups with activity in both human and mouse backgrounds to facilitate animal model engagement for translational development. Chemical modifications within two groups of small molecules produced leads with improved activity over original hits. Furthermore, these leads demonstrated activity in ex vivo cytokine release assays from human blood- and mouse bone marrow-derived macrophages. Dependence on IRF3 was demonstrated using bone marrow-derived macrophages from IRF3-deficient mice, which were not responsive to the molecules. To identify the upstream pathway leading to IRF3 activation, we used a library of CRISPR knockout cell lines to test the key innate immune adaptor and receptor molecules. These studies indicated a surprising toll-interleukin-1 receptor-domain-containing-adapter-inducing interferon-ß-dependent but TLR3/4-independent mechanism of IRF3 activation.

Soluble Epoxide Hydrolase Blockade after Stroke Onset Protects Normal but Not Diabetic Mice.

Soluble epoxide hydrolase (sEH) is abundant in the brain, is upregulated in type 2 diabetes mellitus (DM2), and is possible mediator of ischemic injury via the breakdown of neuroprotective epoxyeicosatrienoic acids (EETs). Prophylactic, pre-ischemic sEH blockade with 4-[[trans-4-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]cyclohexyl]oxy]-benzoic acid (tAUCB) reduces stroke-induced infarct in normal and diabetic mice, with larger neuroprotection in DM2. The present study tested whether benefit occurs in normal and DM2 mice if tAUCB is administered after stroke onset. We performed 60 min middle cerebral artery occlusion in young adult male C57BL mice divided into four groups: normal or DM2, with t-AUCB 2 mg/kg or vehicle 30 min before reperfusion. Endpoints were (1) cerebral blood flow (CBF) by laser Doppler, and (2) brain infarct at 24 h. In nondiabetic mice, t-AUCB reduced infarct size by 30% compared to vehicle-treated mice in the cortex (31.4 ± 4 vs. 43.8 ± 3 (SEM)%, respectively) and 26% in the whole hemisphere (26.3 ± 3 vs. 35.2 ± 2%, both p < 0.05). In contrast, in DM2 mice, tAUCB failed to ameliorate either cortical or hemispheric injury. No differences were seen in CBF. We conclude that tAUCB administered after ischemic stroke onset exerts brain protection in nondiabetic but not DM2 mice, that the neuroprotection appears independent of changes in gross CBF, and that DM2-induced hyperglycemia abolishes t-AUCB-mediated neuroprotection after stroke onset.