Background and Purpose-Premenopausal women are at lower risk than men for stroke, but the comparative vulnerability to tissue injury once a cerebrovascular incident occurs is unknown. We hypothesized that female rats sustain less brain damage than males during experimental focal ischemia and that the gender difference in ischemic outcome can be eliminated by ovariectomy. Methods-Age-matched male (M), intact female (F), and ovariectomized female (O; plasma estradiol: 4.1Ϯ1.6 pg/mL compared with 7.4Ϯ1.5 in F and 4.0Ϯ1.1 in M) rats from two different strains, normotensive Wistar and stroke-prone spontaneously hypertensive rats, were subjected to 2 hours of intraluminal middle cerebral artery occlusion, followed by 22 hours of reperfusion. Cerebral blood flow (CBF) was monitored throughout the ischemic period by laser-Doppler flowmetry. Infarction volume in the cerebral cortex (Ctx) and caudoputamen (CP) was determined by 2,3,5-triphenyltetrazolium chloride staining. In a separate cohort of M, F, and O Wistar rats, absolute rates of regional CBF were measured at the end of the ischemic period by quantitative autoradiography using [ 14 C]iodoantipyrine. Results-F rats of either strain had a smaller infarct size in Ctx and CP and a higher laser-Doppler flow during ischemia compared with respective M and O rats. Mean end-ischemic CBF was higher in F compared with M and O rats in CP, but not in Ctx. Cerebrocortical tissue volume with end-ischemic CBF Ͻ10 mL/100 g/min was smaller in F than M rats, but not different from O rats. Conclusions-We conclude that endogenous estrogen improves stroke outcome during vascular occlusion by exerting both neuroprotective and flow-preserving effects. (Stroke. 1998;29:159-166.)

Background and Purpose-Young adult female rats sustain smaller infarcts after experimental stroke than age-matched males. This sex difference in ischemic brain injury in young animals disappears after surgical ovariectomy and can be restored by estrogen replacement. We sought to determine whether ischemic brain injury continues to be smaller in middle-aged, reproductively senescent female rats compared with age-matched males and to test the effect of ovarian steroids on brain injury after experimental stroke in females. Methods-Four groups of 16-month old Wistar rats (males [nϭ9], untreated females [nϭ9], and females pretreated with 17␤-estradiol [25-g pellets administered subcutaneously for 7 days; nϭ9] or progesterone [10-mg pellets administered subcutaneously for 7 days; nϭ9] were subjected to 2 hours of middle cerebral artery occlusion with the intraluminal filament technique, followed by 22 hours of reperfusion. Physiological variables and laser-Doppler cerebral cortical perfusion were monitored throughout ischemia and early reperfusion. In a separate cohort of males (nϭ3), untreated females (nϭ3), females pretreated with 17␤-estradiol (nϭ3), and females pretreated with progesterone (nϭ3), end-ischemic regional cerebral blood flow was measured by [ 14 C]iodoantipyrine autoradiography. Results-As predicted, infarct size was not different between middle-aged male and female rats. Cortical infarcts were 21Ϯ5% and 31Ϯ6% of ipsilateral cerebral cortex, and striatal infarcts were 44Ϯ7% and 43Ϯ5% of ipsilateral striatum in males and females, respectively. Both estrogen and progesterone reduced cortical infarct in reproductively senescent females (5Ϯ2% and 16Ϯ4% in estrogen-and progesterone-treated groups, respectively, compared with 31Ϯ6% in untreated group). Striatal infarct was smaller in the estrogen-but not in the progesterone-treated group. Relative change in laser-Doppler cerebral cortical perfusion from preischemic baseline and absolute end-ischemic regional cerebral blood flow were not affected by hormonal treatments. Conclusions-We conclude that the protection against ischemic brain injury found in young adult female rats disappears after reproductive senescence in middle-aged females and that ovarian hormones alleviate stroke injury in reproductively senescent female rats by a blood flow-independent mechanism. These findings support a role for hormone replacement therapy in stroke injury prevention in postmenopausal women. (Stroke. 2000;31:161-168.)

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.

Our results demonstrate that a P450 2C11 mRNA is expressed in astrocytes and may be responsible for astrocyte epoxygenase activity. Given the vasodilatory effect of EETs, our findings suggest a role for astrocytes in the control of cerebral microcirculation mediated by P450 2C11-catalyzed conversion of AA to EETs. The mechanism of EET-induced dilation of rat cerebral microvessels may involve activation of K+ channels.

Background and Purpose-The importance of postmenopausal estrogen replacement therapy for stroke in females remains controversial. We previously showed that female rats sustain less infarction in reversible middle cerebral artery occlusion (MCAO) than their ovariectomized counterparts and that vascular mechanisms are partly responsible for improved tissue outcomes. Furthermore, exogenous estrogen strongly protects the male brain, even when administered in a single injection before MCAO injection. The present study examined the hypothesis that replacement of 17␤-estradiol to physiological levels improves stroke outcome in ovariectomized, estrogen-deficient female rats, acting through blood flow-mediated mechanisms. Methods-Age-matched, adult female Wistar rats were ovariectomized and treated with 0, 25, or 100 g of 17␤-estradiol administered through a subcutaneous implant or with a single Premarin (USP) injection (1 mg/kg) given immediately before ischemia was induced (nϭ10 per group). Each animal subsequently underwent 2 hours of MCAO by the intraluminal filament technique, followed by 22 hours of reperfusion. Ipsilateral parietal cortex perfusion was monitored by laser-Doppler flowmetry throughout ischemia. Cortical and caudate-putamen infarction volumes were determined by 2,3,5-triphenyltetrazolium chloride staining and digital image analysis. End-ischemic regional cerebral blood flow was measured in ovariectomized females with 0-or 25-g implants (nϭ4 per group) by 14 C-iodoantipyrine quantitative autoradiography. Results-Plasma estradiol levels were 3.0Ϯ0.6, 20Ϯ8, and 46Ϯ10 pg/mL in the 0-, 25-, and 100-g groups, respectively.Caudate-putamen infarction (% of ipsilateral caudate-putamen) was reduced by long-term, 25-g estrogen treatment (13Ϯ4% versus 31Ϯ6% in the 0-g group, PϽ0.05, and 22Ϯ3% in the 100-g group). Similarly, cortical infarction (% of ipsilateral cortex) was reduced only in the 25-g group (3Ϯ2% versus 12Ϯ3% in the 0-g group, PϽ0.05, and 6Ϯ3% in the 100-g group. End-ischemic striatal or cortical blood flow was not altered by estrogen treatment at the neuroprotective dose. Infarction volume was unchanged by acute treatment before MCAO when estrogen-treated animals were compared with saline vehicle-treated animals. Conclusions-Long-term estradiol replacement within a low physiological range ameliorates ischemic brain injury in previously ovariectomized female rats. The neuroprotective mechanism is flow-independent, not through preservation of residual ischemic regional cerebral blood flow. Furthermore, the therapeutic range is narrow, because the benefit of estrogen in transient vascular occlusion is diminished at larger doses, which yield high, but still physiologically relevant, plasma 17␤-estradiol levels. Lastly, unlike in the male brain, single-injection estrogen exposure does not salvage ischemic tissue in the female brain. Therefore, although exogenous steroid therapy protects both male and female estrogen-deficient brain, the mechanism may not be identical and depends on long-term hormone aug...

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.

Renal cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) regulate sodium transport and blood pressure. Although endothelial CYP-derived EETs are potent vasodilators, their contribution to the regulation of blood pressure remains unclear. Consequently, we developed transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases to increase endothelial EET biosynthesis. Compared to wild-type littermate controls, an attenuated afferent arteriole constrictor response to endothelin-1 and enhanced dilator response to acetylcholine was observed in CYP2J2 and CYP2C8 transgenic mice. CYP2J2 and CYP2C8 transgenic mice demonstrated modestly, but not significantly, lower mean arterial pressure under basal conditions compared to wild-type controls. However, mean arterial pressure was significantly lower in both CYP2J2 and CYP2C8 transgenic mice during coadministration of N-nitro-l-arginine methyl ester and indomethacin. In a separate experiment, a high-salt diet and subcutaneous angiotensin II was administered over 4 wk. The angiotensin/high-salt-induced increase in systolic blood pressure, proteinuria, and glomerular injury was significantly attenuated in CYP2J2 and CYP2C8 transgenic mice compared to wild-type controls. Collectively, these data demonstrate that increased endothelial CYP epoxygenase expression attenuates afferent arteriolar constrictor reactivity and hypertension-induced increases in blood pressure and renal injury in mice. We conclude that endothelial CYP epoxygenase function contributes to the regulation of blood pressure.

Background and Purpose-Estrogen is a known neuroprotective and vasoprotective agent in experimental cerebral ischemia. Preischemic steroid treatment protects animals of both sexes from focal cerebral ischemia. This study determined whether intravenous estrogen acts as a vasodilator when administered on reperfusion and whether the resulting increase in cerebral blood flow (CBF) provides tissue protection from middle cerebral artery occlusion. Methods-Adult male Wistar rats were treated with reversible middle cerebral artery occlusion (2 hours), then infused with intravenous estrogen (Premarin; 1 mg/kg) or vehicle during the first minutes of reperfusion (nϭ15 per group). Cortical laser-Doppler flowmetry was used to assess adequacy of occlusion. Ischemic lesion volume was determined at 22 hours after occlusion by 2,3,5-triphenyltetrazolium chloride staining and image analysis. Cortical and striatal CBF was measured by 14 [C]iodoantipyrine autoradiography at 10 (nϭ10) or 90 (nϭ11) minutes of reperfusion. Results-As expected, supraphysiological plasma estrogen levels were achieved during reperfusion (estrogen, 198Ϯ45 pg/mL; vehicle, 6Ϯ5; Pϭ0.001). Physiological variables were controlled and not different between groups. Total hemispheric infarction was reduced in estrogen-treated rats (estrogen, 49Ϯ4% of ipsilateral structure; vehicle, 33Ϯ5%; Pϭ0.02), which was most pronounced in striatum (estrogen, 40Ϯ6% of ipsilateral striatum; vehicle, 60Ϯ3%; Pϭ0.01). CBF recovery was strikingly increased by estrogen infusion at 10 minutes in frontal (estrogen, 102Ϯ12 mL/100 g per minute; vehicle, 45Ϯ15; Pϭ0.01) and parietal cortex (estrogen, 74Ϯ15 mL/100 g per minute; vehicle, 22Ϯ13; Pϭ0.028) and throughout striatum (estrogen, 87Ϯ13 mL/100 g per minute; vehicle, 25Ϯ20; Pϭ0.02). Hemispheric volume with low CBF recovery (eg, Ͻ20 mL/100 g per minute) was smaller in estrogen-treated animals (estrogen, 73Ϯ18 mm 3 ; vehicle, 257Ϯ46; Pϭ0.002). However, differences in CBF recovery could not be appreciated between groups by 90 minutes of reperfusion. Conclusions-Acute estrogen therapy during reperfusion improves tissue outcome from experimental stroke. The steroid rapidly promotes CBF recovery and reduces hemispheric no-reflow zones. This beneficial effect appears only during early reperfusion and likely complements other known mechanisms by which estrogen salvages brain from focal necrosis. Key Words: cerebral ischemia, focal Ⅲ estrogens Ⅲ gender Ⅲ middle cerebral artery occlusion Ⅲ reperfusion Ⅲ stroke, acute Ⅲ women E strogen has been widely shown to protect brain in numerous models of experimental brain injury. Preischemic steroid treatment has been well studied as a neuroprotective agent in adult animals of both sexes and in reproductively senescent, middle-aged female rats (for reviews, see 1, 2). From the perspective of cerebrovascular disease and stroke, estrogen has been primarily of interest as a postmenopausal hormone therapy that could reduce stroke incidence. The therapeutic utility of the steroid in postischemic treatment p...