Background and Purpose-Cytochrome P450 epoxygenase metabolizes arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs are produced in the brain and perform important biological functions, including vasodilation and neuroprotection. However, EETs are rapidly metabolized via soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs). We tested the hypothesis that sEH gene deletion is protective against focal cerebral ischemia through enhanced collateral blood flow. Methods-sEH knockout (sEHKO) mice with and without EETs antagonist 14, 15 epoxyeicosa-5(Z)-enoic acid (EEZE) were subjected to 2-hour middle cerebral artery occlusion (MCAO), and infarct size was measured at 24 hours of reperfusion and compared to wild-type (WT) mice. Local CBF rates were measured at the end of MCAO using iodoantipyrine (IAP) autoradiography, sEH protein was analyzed by Western blot and immunohistochemistry, and hydrolase activity and levels of EETs/DHETs were measured in brain and plasma using LC-MS/MS and ELISA, respectively. Results-sEH immunoreactivity was detected in WT, but not sEHKO mouse brain, and was localized to vascular and nonvascular cells. 14,15-DHET was abundantly present in WT, but virtually absent in sEHKO mouse plasma. However, hydrolase activity and free 14,15-EET in brain tissue were not different between WT and sEHKO mice. Infarct size was significantly smaller, whereas regional cerebral blood flow rates were significantly higher in sEHKO compared to WT mice. Infarct size reduction was recapitulated by 14,15-EET infusion. However, 14,15-EEZE did not alter infarct size in sEHKO mice. Conclusions-sEH gene deletion is protective against ischemic stroke by a vascular mechanism linked to reduced hydration of circulating EETs.

We previously demonstrated that the neuropeptide cocaine-and amphetamine-regulated transcript (CART) is protective against focal cerebral ischemia in vivo and against neuronal cell death in culture induced by oxygen-glucose deprivation (OGD). The mechanism of neuroprotection by CART is unknown, in part due to lack of knowledge regarding its putative receptor. Using a yeast two-hybrid system with CART's carboxy-terminal to screen a mouse brain cDNA library, we uncovered a potential direct interaction between CART and subunit B of the mitochondrial enzyme succinate dehydrogenase (SDHB). We confirmed CART/SDHB binding using in vitro pull-down assay, and tested the effects of CART peptide on SDH activity, Complex II (CII) activity and ATP production in primary cultured cortical neurons under basal conditions and after OGD. At concentrations between 0.2 and 4 nM, CART significantly increased SDH function, CII activity and ATP generation in purified mitochondria and intact neurons under baseline conditions. Furthermore, pretreatment with CART enhanced mitochondrial mechanisms of neuronal survival and prevented the decline in SDH and CII activities and ATP production after OGD. The findings suggest that CART's neuroprotective mechanism of action may be linked to preservation of mitochondrial function and prevention of energy failure after ischemia-reperfusion injury.

The survival of rat Purkinje cell (PCs) cerebellar cultures was used to test the hypothesis that progesterone is protective againstoxygen-glucose deprivation through potentiation of GABA A receptor activity. Electrophysiological recordings confirm that PCs develop robust excitatory and inhibitory synapses in culture. Exposure of cultured PCs to increasing concentrations of progesterone during oxygen-glucose deprivation revealed a concentration-dependent protection by progesterone, with significant protection observed at physiological concentrations, as low as 10 nm. The concurrent application of the GABA A receptor antagonist picrotoxin (100 µm) completely abolished the neuroprotection afforded by progesterone, indicating that progesterone is neuroprotective through activation of GABA A receptors. Progesterone potentiates GABA A receptor activity indirectly through its metabolites, such as allopregnanolone (ALLO). Therefore, ALLO was applied to PC cultures and was observed to produce significant protection at all concentrations tested, from 10 to 1000 nm. Finally, the inhibition of progesterone metabolism with finasteride abolished the protection afforded by progesterone without having any effect on the neuroprotection caused by ALLO. These data indicate that progesterone protects cerebellar PCs at physiological concentrations through a GABA-active metabolite.

Cerebellar Purkinje cells (PC) are particularly vulnerable to ischemic injury and excitotoxicity, although the molecular basis of this sensitivity remains unclear. We tested the hypothesis that ischemia causes rapid down‐regulation of GABAA receptors in cerebellar PC, thereby increasing susceptibility to excitotoxicity. Oxygen‐glucose deprivation (OGD) caused a decline in functional GABAA receptors, within the first hour of re‐oxygenation. Decreased amplitude of miniature inhibitory post‐synaptic potentials confirmed that OGD caused a significant decrease in functional synaptic GABAA receptors and quantitative Western blot analysis demonstrated the loss of GABAA receptor current was associated with a decline in total receptor protein. Interestingly, the potent neuroprotectant allopregnanolone (ALLO) prevented the decline in GABAA receptor current and protein. Consistent with our in vitro data, global ischemia in mice caused a significant decline in total cerebellar GABAA receptor protein and PC specific immunoreactivity. Moreover, ALLO provided strong protection of PC and prevented ischemia‐induced decline in GABAA receptor protein. Our findings indicate that ischemia causes a rapid and sustained loss of GABAA receptors in PC, whereas ALLO prevents the decline in GABAA receptors and protects against ischemia‐induced damage. Thus, interventions which prevent ischemia‐induced decline in GABAA receptors may represent a novel neuroprotective strategy.

Activation of poly (ADP-ribose) polymerases contributes to ischemic damage by causing neuronal NAD+ depletion, release of apoptosis-inducing factor and consequent caspase-independent cell death. PARP-mediated cell death is sexually dimorphic, participating in ischemic damage in the male brain, but not the female brain. We tested the hypothesis that androgen signaling is required for this male-specific neuronal cell death pathway. We observed smaller damage following focal cerebral ischemia (MCAO) in male PARP-1 knockout mice compared to WT as well as decreased damage in male mice treated with the PARP inhibitor PJ34. Protection from ischemic damage provided by PJ-34 in WT mice is lost after removal of testicular androgens (CAST) and rescued by androgen replacement. CAST PARP-1 KO mice exhibit increased damage compared to intact male KO mice, an effect reversed by androgen replacement in an androgen receptor-dependent manner. Lastly, we observed that ischemia causes an increase in PARP-1 expression that is diminished in the absence of testicular androgens. Our data indicates that PARP-mediated neuronal cell death in the male brain requires intact androgen-androgen receptor signaling.