The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are produced in brain and perform important biological functions, including protection from ischemic injury. The beneficial effect of EETs, however, is limited by their metabolism via soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH inhibition is protective against ischemic brain damage in vivo by a mechanism linked to enhanced cerebral blood flow (CBF). We determined expression and distribution of sEH immunoreactivity (IR) in brain, and examined the effect of sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) on CBF and infarct size after experimental stroke in mice. Mice were administered a single intraperitoneal injection of AUDA-BE (10 mg/kg) or vehicle at 30 mins before 2-h middle cerebral artery occlusion (MCAO) or at reperfusion, in the presence and absence of P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH). Immunoreactivity for sEH was detected in vascular and non-vascular brain compartments, with predominant expression in neuronal cell bodies and processes. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester was detected in plasma and brain for up to 24 h after intraperitoneal injection, which was associated with inhibition of sEH activity in brain tissue. Finally, AUDA-BE significantly reduced infarct size at 24 h after MCAO, which was prevented by MS-PPOH. However, regional CBF rates measured by iodoantipyrine (IAP) autoradiography at end ischemia revealed no differences between AUDA-BE- and vehicle-treated mice. The findings suggest that sEH inhibition is protective against ischemic injury by non-vascular mechanisms, and that sEH may serve as a therapeutic target in stroke.

Microglia are resident CNS immune cells that are active sensors in healthy brain and versatile effectors under pathological conditions. Cerebral ischemia induces a robust neuroinflammatory response that includes marked changes in the gene-expression profile and phenotype of a variety of endogenous CNS cell types (astrocytes, neurons and microglia), as well as an influx of leukocytic cells (neutrophils, macrophages and T-cells) from the periphery. Many molecules and conditions can trigger a transformation of surveying microglia to microglia of an alerted or reactive state. Here we review recent developments in the literature that relate to microglial activation in the experimental setting of in vitro and in vivo ischemia. We also present new data from our own laboratory demonstrating the direct effects of in vitro ischemic conditions on the microglial phenotype and genomic profile. In particular, we focus on the role of specific molecular signaling systems, such as hypoxia inducible factor-1 and Toll-like receptor-4, in regulating the microglial response in this setting. We then review histological and novel radiological data that confirm a key role for microglial activation in the setting of ischemic stroke in humans. We also discuss recent progress in the pharmacologic and molecular targeting of microglia in acute ischemic stroke. Finally, we explore how recent studies on ischemic preconditioning have increased interest in pre-emptively targeting microglial activation in order to reduce stroke severity.

Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine-and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases.ischemia ͉ stroke ͉ estrogen

Single nucleotide polymorphisms (SNPs) in the human EPHX2 gene have recently been implicated in susceptibility to cardiovascular disease, including stroke. EPHX2 encodes for soluble epoxide hydrolase (sEH), an important enzyme in the metabolic breakdown of arachidonic acid-derived eicosanoids referred to as epoxyeicosatrienoic acids (EETs). We previously demonstrated that EETs are protective against ischemic cell death in culture. Therefore, we tested the hypothesis that polymorphisms in the human EPHX2 gene alter sEH enzyme activity and affect neuronal survival after ischemic injury in vitro. Human EPHX2 mutants were recreated by site-directed mutagenesis and fused downstream of TAT protein transduction domain. Western blot analysis and immunocytochemistry staining revealed high-transduction efficiency of human TAT-sEH variants in rat primary cultured cortical neurons, associated with increased metabolism of 14,15-EET to corresponding 14,15-dihydroxyeicosatrienoic acid. A human variant of sEH with Arg103Cys amino acid substitution, previously demonstrated to increase sEH enzymatic activity, was associated with increased cell death induced in cortical neurons by oxygen-glucose deprivation (OGD) and reoxygenation. In contrast, the Arg287Gln mutation was associated with reduced sEH activity and protection from OGD-induced neuronal cell death. We conclude that sequence variations in the human EPHX2 gene alter susceptibility to ischemic injury and neuronal survival in a manner linked to changes in the hydrolase activity of the enzyme. The findings suggest that human EPHX2 mutations may in part explain the genetic variability in sensitivity to ischemic brain injury and stroke outcome.

Biologic sex and sex steroids are important factors in clinical and experimental stroke and traumatic brain injury (TBI). Laboratory data strongly show that progesterone treatment after TBI reduces edema, improves outcomes and restores blood brain barrier function. Clinical studies to date agree with these data, and there are ongoing human trials for progesterone treatment after TBI. Estrogen has accumulated an impressive reputation as a neuroprotectant when evaluated at physiologically relevant doses in laboratory studies of stroke, but translation to patients remains to be shown. The role of androgens in male stroke or TBI is understudied and important to pursue given the epidemiology of stroke and trauma in men. To date, male sex steroids remain largely evaluated at the bench rather than the bedside. This review evaluates key evidence and highlights the importance of the platform on which brain injury occurs, i.e. genetic sex and hormonal modulators. Keywordsstroke; brain ischemia; estradiol; neurosteroids; progesterone; allopreganolone; androgens Sex differences in brain injuryIt is now clear that biological sex alters the incidence of, and outcome from, ischemic and traumatic brain injury. For example, male sex is an acknowledged risk factor for stroke, and in most epidemiological series, stroke occurs more frequently in men vs. women. This sexually dimorphic disease pattern remains apparent until ages well beyond the menopausal years 1, 2 . Nevertheless, stroke risk increases with age in both sexes, and there is broad evidence that outcome from an ischemic event is worse in aged women than in their male counterparts. Knowledge of mechanisms of ischemic cell death and neuroprotection is important for both sexes, but current evidence suggests that these mechanisms are not identical in males and females. a. Sex differences in ischemic outcomes: animal modelsAnimal models of brain injury, typically rodents, have been used to evaluate side-by-side outcomes from ischemia or trauma. In most reports, females fare better than do their agematched male counterparts. Early evidence in female vs. male spontaneously hypertensive, genetically stroke prone rats uncovered the male phenotype of "ischemia-sensitivity" 3 . This landmark study of 2000 animals showed that life expectancy is longer in the female, and the development of cerebral hemorrhage is delayed until an advanced age 3 . Subsequently, a In summary, these findings suggest that the response to cerebral injury in vivo and in vitro is partially a function of the sex of the cell. However, this in no way discounts the importance of gonadal steroids or brain-derived neurosteroids as modulators of oxidant, toxic and ischemic challenges to the brain. The role of progesterone, estradiol and testosterone in shaping neuroinjury is reviewed in subsequent sections. Neuroprotective effects of ProgesteroneProgesterone is synthesized from cholesterol by the gonads, adrenal gland or placenta. In addition, progesterone can be generated within the brain as a neurosteroi...