Recombinant T-cell receptor ligand RTL1000 limits inflammation and decreases infarct size after experimental ischemic stroke in middle-aged mice.

We have previously demonstrated that recombinant T-cell receptor ligand 1000 (RTL1000) reduces infarct size and improves long-term functional recovery after experimental stroke in young transgenic mice expressing human leukocyte antigen DR2 (DR2-Tg). In this study, we determined the effect of RTL1000 on infarct size in 12-month-old middle-aged DR2-Tg mice, and investigated its mechanism of action. Twelve-month-old male DR2-Tg mice underwent 60min of intraluminal reversible middle cerebral artery occlusion (MCAO). Vehicle or RTL1000 was injected 4, 24, 48 and 72h after MCAO. Cortical, striatal and total hemispheric infarcts were measured 96h after stroke. Spleen and brain tissues were collected 96h after stroke for immunological analysis. Our data showed that RTL1000 significantly reduced infarct size 96h after MCAO in middle-aged male DR2-Tg mice. RTL1000 decreased the number of activated monocytes/microglia cells (CD11b(+)CD45(hi)) and CD3(+) T cells in the ischemic hemisphere. RTL1000 also reduced the percentage of total T cells and inflammatory neutrophils in the spleen. These findings suggest that RTL1000 protects against ischemic stroke in middle-aged male mice by limiting post-ischemic inflammation.

Delayed administration of a PTEN inhibitor BPV improves functional recovery after experimental stroke.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitors administered prior to or immediately after experimental stroke confer acute neuroprotection. However, it remains unclear if delayed treatment with a PTEN inhibitor improves long-term functional recovery after stroke. We addressed the issue in this study. Adult male mice were subjected to 1h of middle cerebral artery occlusion (MCAO) followed by treatment with a well-established PTEN inhibitor BPV or saline daily for 14 days, starting at 24h after MCAO. Functional recovery was assessed with behavioral tests and acute infarct volumes were analyzed histologically. Delayed BPV treatment did not reduce infarction during the acute phase, but significantly improved long-term functional recovery after MCAO. Since PTEN is a critical intrinsic inhibitory factor in axonal regeneration, we further examined BPV effects on axonal densities following MCAO using bielschowsky silver staining and immunohistochemistry with antibodies against myelin basic protein. Delayed BPV treatment significantly increased axon densities in the ischemic brain at 14 days after MCAO. Moreover, PTEN expression persistently remained high in the ischemic brain over 14 days after MCAO, and BPV treatment increased post-ischemic activation of Akt and mTOR in the ischemic brain. Akt and mTOR activation are the well-established cascades downstream to PTEN inhibition and have been shown to contribute to post-injury axonal regrowth in response to PTEN inhibition. Consistently, in an in vitro neuronal ischemia model, BPV enhanced axonal outgrowth of primary cortical neurons after oxygen-glucose deprivation and the enhancing effects were abolished by Akt/mTOR inhibition. In conclusion, delayed BPV treatment improved functional recovery from experimental stroke possibly via enhancing axonal growth and Akt/mTOR activation contributed to BPV-enhanced post-stroke axon growth.

Mechanism of the sex difference in neuronal ischemic cell death.

BACKGROUND: Stroke risk and outcome are different in men and women. We hypothesized that this is partly due to an inherent difference in susceptibility to ischemia between neurons from male vs. female brains. We tested whether neurons from male rodents are more susceptible to in-vitro ischemia than cells from females, and if this is related to increased expression of soluble epoxide hydrolase (sEH). sEH contributes to neuronal cell death by inactivating neuroprotective epoxyeicosatrienoic acids (EETs). METHODS: Rodent cortical neurons were cultured, and exposed to oxygen-glucose deprivation (OGD); then cell death was measured. EETs levels were determined by LC-MS/MS. Expression of sEH-encoding ephx2 was determined by qRT-PCR. Western blotting, immunocytochemistry, and hydrolase activity assay assessed protein expression and activity. RESULTS: Cell death after OGD was higher in neurons from males vs. females, which correlated with higher ephx2 mRNA and stronger sEH immunoreactivity. However, EETs levels were similar in both sexes and pharmacological inhibition of the hydrolase domain of sEH did not abolish the sex difference in cell death. Genetic knockout of sEH in mice abolished the sex difference observed in neurons isolated from these mice after OGD. CONCLUSIONS: Cultured cortical neurons from females are more resistant to ischemia than neurons from males. Neurons from females have less sEH activity compared to neurons from males at baseline, although sEH levels were not measured after OGD. While pharmacological inhibition of the hydrolase domain of sEH does not affect cell death, knockout of the gene encoding sEH eradicates the sex difference seen in wild-type neurons, suggesting a role for further study of the lesser-known phosphatase domain of sEH and its role in sexual dimorphism in neuronal sensitivity to ischemia.

Brainstem control of cerebral blood flow and application to acute vasospasm following experimental subarachnoid hemorrhage.

Symptomatic ischemia following aneurysmal subarachnoid hemorrhage (SAH) is common but poorly understood and inadequately treated. Severe constriction of the major arteries at the base of the brain, termed vasospasm, traditionally has been thought to be a proximal event underlying these ischemias, although microvascular changes also have been described. The vast majority of studies aimed at understanding the pathogenesis of ischemic deficits, and vasospasm have focused on the interaction of the "spasmogen" of the extravasated blood with the smooth muscle and endothelium of the arteries. This has led to a comparative neglect of the contribution of the CNS to the maintenance of cerebral perfusion. In the present study, we focused on the role of the rostral ventromedial medulla (RVM) in modulating cerebral perfusion at rest and following an experimental SAH in the rat. Changes in cerebral blood flow (CBF) were measured using laser-Doppler flowmetry and three-dimensional optical microangiography. Focal application of a GABA(A) receptor agonist and antagonist was used to respectively inactivate and activate the RVM. We show here that the RVM modulates cerebral blood flow under resting conditions, and further, contributes to restoration of cerebral perfusion following a high-grade SAH. Failure of this brainstem compensatory mechanism could be significant for acute perfusion deficits seen in patients following subarachnoid hemorrhage.

Cytochrome P450 in neurological disease.

Advances in a multitude of disciplines support an emerging role for cytochrome P450 enzymes and their metabolic substrates and end-products in the pathogenesis and treatment of central nervous system disorders, including acute cerebrovascular injury, such as stroke, chronic neurodegenerative disease, such as Alzheimer's and Parkinson's disease, as well as epilepsy, multiple sclerosis and psychiatric disorders, including anxiety and depression. The neural tissue contains its own unique set of P450 genes that are regulated in a manner that is distinct from their molecular regulation in peripheral tissue. Furthermore, brain P450s catalyze the formation of important brain signaling molecules, such as neurosteroids and eicosanoids, and metabolize substrates as diverse as vitamins A and D, cholesterol, bile acids, as well as centrally acting drugs, anesthetics and environmental neurotoxins. These unique characteristics allow this family of proteins and their metabolites to perform such vital functions in brain as neurotrophic support, neuroprotection, control of cerebral blood flow, temperature control, neuropeptide release, maintenance of brain cholesterol homoeostasis, elimination of retinoids from CNS, regulation of neurotransmitter levels and other functions important in brain physiology, development and disease.

Social stress exacerbates stroke outcome by suppressing Bcl-2 expression.

The relationship between stressful life events and the onset of disease is well documented. However, the role of psychological stress as a risk factor for life-threatening cerebrovascular insults such as stroke remains unspecified, but could explain individual variation in stroke outcome. To discover the mechanisms through which psychological stress may alter stroke outcome, we modeled the effects of chronic social intimidation and stress on ischemia-induced bcl-2 expression and early neuronal cell loss resulting from cerebral artery occlusion in mice (C57BL/6). The bcl-2 protooncogene promotes cell survival and protects against apoptosis and cellular necrosis in numerous neurodegenerative disorders, including stroke. In our study, male mice were chronically exposed to aggressive social stimuli before induction of a controlled, mild ischemic insult. Stressed mice expressed approximately 70% less bcl-2 mRNA than unstressed mice after ischemia. In addition, social stress greatly exacerbated infarct in wild-type mice but not in transgenic mice that constitutively express increased neuronal bcl-2. Despite similar postischemic concentrations of corticosterone, the major stress hormone in mice, high corticosterone concentrations were significantly correlated with larger infarcts in wild-type mice but not bcl-2 transgenic mice. Thus, enhanced bcl-2 expression offsets the potentially deleterious consequences of high postischemic plasma corticosterone concentrations. Taken together, these data demonstrate that stressful prestroke social milieu strongly compromises an endogenous molecular mechanism of neuroprotection in injured brain and offer a new behavioral target for stroke therapy.

Estrogen and Bcl-2: gene induction and effect of transgene in experimental stroke.

Female rodents producing endogenous estrogens are protected from stroke damage in comparison with male counterparts. This natural protection is lost after ovariectomy or reproductive senescence. The aim of this study is to determine whether estrogen reduces early neuronal injury and cell loss after ischemia by increasing the expression of Bcl-2. Male, intact female, ovariectomized, and estrogen-repleted ovariectomized rats were subjected to middle cerebral artery occlusion, and 22 hr later the level and localization of Bcl-2 mRNA and protein were determined. The levels of post-ischemic bcl-2 mRNA and protein were increased exclusively in neurons within the peri-infarct region. Intact females and estrogen-treated castrates demonstrated increased bcl-2 mRNA and protein expression compared with males and estrogen-deficient females, accompanied by a decrease in infarct size. To test the hypothesis that the neuroprotective mechanism of estrogen functions via Bcl-2, we compared ischemic outcome in male, female, and ovariectomized wild-type mice and mice overexpressing Bcl-2 exclusively in neurons. Wild-type female mice sustained smaller infarcts compared with males. Bcl-2 overexpression reduced infarct size in males, but provided no added protection in the female. Moreover, ovariectomy exacerbated infarction in wild-type females, but had no effect in Bcl-2 overexpressors. These data indicate that overexpression of Bcl-2 simulates the protection against ischemic injury conferred by endogenous female sex steroids. We concluded that estrogen rescues neurons after focal cerebral ischemia by increasing the level of Bcl-2 in peri-infarct regions and that estrogen-induced bcl-2 gene expression is an important downstream component of neuronal protection in female stroke.

Anesthetic choice of halothane versus propofol: impact on experimental perioperative stroke.

BACKGROUND AND PURPOSE: It is not known whether preischemic exposure to anesthetic agents affects the amount of damage from transient focal ischemia that occurs after cessation of the anesthetic. We compared the effect of prior exposure to halothane or propofol on infarction size after transient middle cerebral artery occlusion (MCAO) induced in the awakening animal to test the hypothesis that anesthetic type and exposure duration would independently affect the amount of brain injury. METHODS: Male Wistar rats (weight, 200 to 300 g) were anesthetized briefly with halothane for placement of hemodynamic instrumentation. Twenty-four hours later, rats were treated with either a short (approximately 1 hour) or long (8 hours) duration of inhaled halothane (1% to 2%) or intravenous propofol (10 mg/kg bolus, 30 mg/kg per hour infusion). Each cohort (n=8 per group) was then subjected to 2-hour MCAO by the intraluminal suture technique. All anesthesia was discontinued once MCAO was achieved. Infarct volume was measured at 22 hours of reperfusion. In a second cohort, regional cerebral blood flow (CBF) was measured ([(14)C]iodoantipyrine autoradiography) at end-occlusion in short-duration halothane (n=5) or short-duration propofol (n=5) anesthesia groups and in corresponding surgical shams (n=3 each). RESULTS: Pericranial temperature, PaO(2), PaCO(2), and blood pressure were controlled and not different among groups before or during occlusion. MCAO resulted in a similar immediate reduction in laser-Doppler flow signal after discontinuation of anesthesia in the awakening animals. Infarct volume was smaller in rats exposed to short-duration halothane in cortex (87.5+/-16.6 mm(3)) (mean+/-SEM) and caudoputamen (38.3+/-13.7 mm(3)) compared with rats exposed to short-duration propofol (cortex, 177.5+/-16.9 mm(3); caudoputamen, 47.8+/-2.9 mm(3)). Infarct volume was not different in long-duration halothane versus long-duration propofol treatment. Absolute cortical or caudoputamen intraischemic CBF was not different between short-duration halothane or short-duration propofol treatment. CONCLUSIONS: These data demonstrate that short-duration halothane exposure before MCAO in the awakening animal attenuates infarction volume compared with propofol. This protection by halothane is not mediated through preservation of intraischemic CBF. Longer durations of halothane exposure may activate secondary injury pathways, which negate the protective effects of short-term halothane preischemic treatment.

Postischemic estrogen reduces hypoperfusion and secondary ischemia after experimental stroke.

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 (n=15 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 (n=10) or 90 (n=11) minutes of reperfusion. RESULTS: As expected, supraphysiological plasma estrogen levels were achieved during reperfusion (estrogen, 198+/-45 pg/mL; vehicle, 6+/-5; P:=0.001). Physiological variables were controlled and not different between groups. Total hemispheric infarction was reduced in estrogen-treated rats (estrogen, 49+/-4% of ipsilateral structure; vehicle, 33+/-5%; P:=0.02), which was most pronounced in striatum (estrogen, 40+/-6% of ipsilateral striatum; vehicle, 60+/-3%; P:=0.01). CBF recovery was strikingly increased by estrogen infusion at 10 minutes in frontal (estrogen, 102+/-12 mL/100 g per minute; vehicle, 45+/-15; P:=0.01) and parietal cortex (estrogen, 74+/-15 mL/100 g per minute; vehicle, 22+/-13; P:=0.028) and throughout striatum (estrogen, 87+/-13 mL/100 g per minute; vehicle, 25+/-20; P:=0.02). Hemispheric volume with low CBF recovery (eg, <20 mL/100 g per minute) was smaller in estrogen-treated animals (estrogen, 73+/-18 mm(3); vehicle, 257+/-46; P:=0.002). 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.

Experimental stroke in the female diabetic, db/db, mouse.

Diabetic hyperglycemia increases brain damage after cerebral ischemia in animals and humans, although the underlying mechanisms remain unclear. Gender-linked differences in ischemic tolerance have been described but have not been studied in the context of diabetes. In the current study, we used a model of unilateral common carotid artery ligation, combined with systemic hypoxia, to study the effects of diabetes and gender on hypoxic-ischemic (HI) brain damage in the genetic model of Type II diabetes, the db/db, mouse. Male and female, control and db/db, mice were subjected to right common carotid artery ligation followed by varying periods of hypoxia (8% oxygen/92% nitrogen) to assess mortality, infarct volume, and tissue damage by light microscopic techniques. End-ischemic regional cerebral blood flow (CBF) was determined using [14C] iodoantipyrine autoradiography. Glycolytic and high energy phosphate compounds were measured in blood and brain by enzymatic and fluorometric techniques. Gender and diabetes had significant effects on mortality from HI and extent of brain damage in the survivors. Female mice were more resistant than their male counterparts, such that the severity (mortality and infarction size) in the male diabetics > female diabetics - male controls > female controls. Endischemic CBF and depletion of cerebral high energy reserves were comparable among all groups. Surprisingly, female diabetic mice were more hyperglycemic and demonstrated a greater prolonged lactacidosis than the males; however, they were more resistant to damage. The results suggest a unique pathophysiology of hypoxia-ischemia in the female diabetic brain.

Hypertonic saline worsens infarct volume after transient focal ischemia in rats.

BACKGROUND AND PURPOSE: Hypertonic saline (HS) has been advocated as a hyperosmolar agent for the treatment of cerebral edema, especially after traumatic brain injury. We tested the hypothesis that continuous intravenous HS administered during reperfusion from transient focal cerebral ischemia attenuates infarct volume. METHODS: Halothane-anesthetized male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. At the onset of reperfusion, rats received a 10-mL/kg intravenous bolus of 0.9% saline (SAL, n=8) or 7.5% SAL (chloride:acetate 50:50, n=8) followed by a continuous infusion for 22 hours. In a second series of experiments, ischemic damage was determined in cohorts treated with equivolumetric 3% saline (n=8) or 20% mannitol (n=8). In a third series, regional cerebral blood flow was measured ([(14)C]iodoantipyrine autoradiography) at 6 hours of reperfusion in 7.5%-SAL-treated (n=5) or SAL-treated (n=5) animals. RESULTS: In SAL rats, serum Na(+) was 137+/-3 and 138+/-2 mEq/L (mean+/-SEM) at baseline and 22 hours of reperfusion, respectively. In 7.5% SAL, serum Na(+) was 136+/-2 and 154+/-2 mEq/L at baseline and reperfusion, respectively. Physiological variables and reduction in laser-Doppler signal during MCAO and early reperfusion were not different between the 2 treatment groups. Cortical infarct volume was larger in 7.5%-SAL-treated rats (121+/-14 mm(3); 30+/-3% of contralateral cortex; P<0.05) than in SAL (64+/-15 mm(3); 16+/-4% of contralateral cortex). Striatal infarct volume was unchanged by HS therapy. Ipsilateral cortical tissue volume was increased relative to the contralateral side (by 26+/-5% with SAL; by 41+/-5% with 7.5% SAL). In contrast, ischemic damage was unaffected by 3%-SAL or 20%-mannitol treatment compared with SAL. Regional cerebral blood flow during reperfusion was heterogeneous in all animals, but there was no evidence of postischemic hypoperfusion or blood flow maldistribution in 7.5%-SAL-treated animals. CONCLUSIONS: These data demonstrate that hypernatremia resulting from postischemic HS infusion worsens cortical infarct volume in transient focal cerebral ischemia. The deleterious effect is not linked to exacerbation of delayed hypoperfusion during early reperfusion (6 hours); however, blood flow defects at later recovery time points remain to be excluded. These results may have implications for HS therapy in clinical ischemic stroke.

Stroke in estrogen receptor-alpha-deficient mice.

BACKGROUND AND PURPOSE: Recent evidence suggests that endogenous estrogens or hormone replacement therapy can ameliorate brain damage from experimental stroke. Protective mechanisms involve enhanced cerebral vasodilation during ischemic stress as well as direct preservation of neuronal viability. We hypothesized that if the intracellular estrogen receptor subtype-alpha (ERalpha) is important to estrogen's signaling in the ischemic brain, then ERalpha-deficient (knockout) (ERalphaKO) female mice would sustain exaggerated cerebral infarction damage after middle cerebral artery occlusion. METHODS: The histopathology of cresyl violet-stained tissues was evaluated after reversible middle cerebral artery occlusion (2 hours, followed by 22 hours of reperfusion) in ERalphaKO transgenic and wild-type (WT) mice (C57BL/6J background strain). End-ischemic cerebral blood flow mapping was obtained from additional female murine cohorts by using [(14)C]iodoantipyrine autoradiography. RESULTS: Total hemispheric tissue damage was not altered by ERalpha deficiency in female mice: 51.9+/-10.6 mm(3) in ERalphaKO versus 60.5+/-5.0 mm(3) in WT. Striatal infarction was equivalent, 12.2+/-1.7 mm(3) in ERalphaKO and 13.4+/-1.0 mm(3) in WT mice, but cortical infarction was paradoxically smaller relative to that of the WT (20.7+/-4.5 mm(3) in ERalphaKO versus 30.6+/-4.1 mm(3) in WT). Intraocclusion blood flow to the parietal cortex was higher in ERalphaKO than in WT mice, likely accounting for the reduced infarction in this anatomic area. There were no differences in stroke outcomes by region or genotype in male animals. CONCLUSIONS: Loss of ERalpha does not enhance tissue damage in the female animal, suggesting that estrogen inhibits brain injury by mechanisms that do not depend on activation of this receptor subtype.

Is the acetazolamide test valid for quantitative assessment of maximal cerebral autoregulatory vasodilation? An experimental study.

BACKGROUND AND PURPOSE: The cerebral vasodilating effect of acetazolamide (ACZ) injection has been used as an index of the autoregulatory vasodilation (or cerebral perfusion reserve). The question of whether the ACZ test assesses the maximal autoregulatory vasodilating capacity is not definitely resolved. The effects of ACZ injection on this reserve at a dose producing maximal vasodilation have never been evaluated and may help to resolve this problem. METHODS: The effect of ACZ injection on cerebral blood flow (CBF) autoregulation was tested in anesthetized rats. A pilot experiment evaluated the dose-effect relationship of injected ACZ, cumulative doses (n=4, group 1), and independent bolus doses (n=6, group 2). CBF was estimated by laser-Doppler flowmetry, and cerebrovascular resistance (CVR) was calculated from mean arterial blood pressure (MABP) and from CBF (expressed as a percentage of baseline CBF). A bolus of ACZ of 21 mg/kg produced the maximal cerebral vasodilation that could be obtained by ACZ administration. In the main experiment, MABP was lowered from 110 to 20 mm Hg by stepwise bleeding in 3 groups of 6 animals treated 10 minutes before bleeding by injection of saline (group 3), 7 mg/kg ACZ (group 4), or 21 mg/kg ACZ (group 5). RESULTS: The CVR-MABP relationship was linear in all groups, indicating that CBF autoregulation was still effective after ACZ administration. CONCLUSIONS: These results indicate that maximal ACZ-induced cerebral vasodilation is not quantitatively equivalent to maximal autoregulatory vasodilating capacity in anesthetized rats.

Estrogen receptor antagonist ICI182,780 exacerbates ischemic injury in female mouse.

Recent findings in animals emphasize that experimental ischemic brain damage can be strikingly reduced by estrogen: however, the neuroprotective mechanisms are not well understood. It was hypothesized that estrogen signaling via cognate estrogen receptors (ERs) within the vasculature is an important aspect of cerebral ischemic protection in the female brain, in part by amplifying intraischemic cerebral blood flow (CBF). In the present study, the hypothesis that chronic treatment with the pure ER antagonist ICI182,780 (ICI) would increase ischemic brain damage by a blood flow-mediated mechanism was investigated. Adult C57B1/6J mice were pretreated with either subcutaneous ICI (100 microg/day) or oil/ethanol vehicle for 1 week before 2 hours of middle cerebral artery occlusion (MCAO) and 22 hours of reperfusion. End-ischemic regional CBF was evaluated in additional cohorts using [14C]iodoantipyrine autoradiography. Infarction volume as measured by cresyl violet histology was greater in the striatum of ICI-treated females (70 +/- 3% of contralateral striatum vs. 40 +/- 12% in vehicle-treated females). Cortical injury was not enhanced relative to control animals (39 +/- 6% of contralateral cortex in ICI group vs. 27 +/- 8% in vehicle-treated group). Physiologic variables and ischemic reduction of the ipsilateral cortical laser-Doppler flow signal were similar between groups. Further, ICI treatment did not alter end-ischemic cortical or striatal CBF. The deleterious effect of ICI was limited to females, as there were no differences in stroke damage or CBF between male treatment groups. These data suggest that estrogen inhibits ischemic brain injury in striatum of the female by receptor-mediated mechanisms that are not linked to preservation of intraischemic CBF.

Neuroprotective effects of female gonadal steroids in reproductively senescent female rats.

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 [n=9], untreated females [n=9], and females pretreated with 17beta-estradiol [25-microgram pellets administered subcutaneously for 7 days; n=9] or progesterone [10-mg pellets administered subcutaneously for 7 days; n=9] 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 (n=3), untreated females (n=3), females pretreated with 17beta-estradiol (n=3), and females pretreated with progesterone (n=3), 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 21+/-5% and 31+/-6% of ipsilateral cerebral cortex, and striatal infarcts were 44+/-7% and 43+/-5% of ipsilateral striatum in males and females, respectively. Both estrogen and progesterone reduced cortical infarct in reproductively senescent females (5+/-2% and 16+/-4% in estrogen- and progesterone-treated groups, respectively, compared with 31+/-6% 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.

Heme oxygenase-2 is neuroprotective in cerebral ischemia.

Heme oxygenase (HO) is believed to be a potent antioxidant enzyme in the nervous system; it degrades heme from heme-containing proteins, giving rise to carbon monoxide, iron, and biliverdin, which is rapidly reduced to bilirubin. The first identified isoform of the enzyme, HO1, is an inducible heat-shock protein expressed in high levels in peripheral organs and barely detectable under normal conditions in the brain, whereas HO2 is constitutive and most highly concentrated in the brain. Interestingly, although HO2 is constitutively expressed, its activity can be modulated by phosphorylation. We demonstrated that bilirubin, formed from HO2, is neuroprotectant, as neurotoxicity is augmented in neuronal cultures from mice with targeted deletion of HO2 (HO2(-/-)) and reversed by low concentrations of bilirubin. We now show that neural damage following middle cerebral artery occlusion (MCAO) and reperfusion, a model of focal ischemia of vascular stroke, is substantially worsened in HO2(-/-) animals. By contrast, stroke damage is not significantly altered in HO1(-/-) mice, despite their greater debility. Neural damage following intracranial injections of N-methyl-d-aspartate (NMDA) is also accentuated in HO2(-/-) animals. These findings establish HO2 as an endogenous neuroprotective system in the brain whose pharmacologic manipulation may have therapeutic relevance.

17beta-estradiol reduces stroke injury in estrogen-deficient female animals.

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 17beta-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 microgram of 17beta-estradiol administered through a subcutaneous implant or with a single Premarin (USP) injection (1 mg/kg) given immediately before ischemia was induced (n=10 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-microgram implants (n=4 per group) by (14)C-iodoantipyrine quantitative autoradiography. RESULTS: Plasma estradiol levels were 3.0+/-0.6, 20+/-8, and 46+/-10 pg/mL in the 0-, 25-, and 100-microgram groups, respectively. Caudate-putamen infarction (% of ipsilateral caudate-putamen) was reduced by long-term, 25-microgram estrogen treatment (13+/-4% versus 31+/-6% in the 0-microgram group, P<0.05, and 22+/-3% in the 100-microgram group). Similarly, cortical infarction (% of ipsilateral cortex) was reduced only in the 25-microgram group (3+/-2% versus 12+/-3% in the 0-microgram group, P<0.05, and 6+/-3% in the 100-microgram 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 17beta-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 augmentation in the female.

Gender-linked brain injury in experimental stroke.

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: 4.1+/-1.6 pg/mL compared with 7.4+/-1.5 in F and 4.0+/-1.1 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 [14C]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 < 10 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.

Functional hyperemia in the brain: hypothesis for astrocyte-derived vasodilator metabolites.

BACKGROUND: Cerebral blood flow is tightly coupled to neuronal metabolic activity, a phenomenon referred to as functional hyperemia. The mechanisms underlying functional hyperemia in the brain have been extensively studied, but the link between neuronal activation and nutritive blood flow has yet to be defined. Recent investigations by our laboratory and others have identified a potential role for astrocytes as an intermediary cell type in this process. SUMMARY OF REVIEW: This short review will develop the hypothesis that cytochrome P450 epoxygenase activity in astrocytes catalyzes formation of epoxyeicosatrienoic acids (EETs), which act as potent dilators of cerebral vessels and are released in response to glutamate receptor activation within astrocytes. Neuronal activity stimulates release of arachidonic acid from the phospholipid pool of astrocytic membranes. We provide evidence that the arachidonic acid released on stimulation of glutamate receptors within astrocytes is metabolized by cytochrome P450 2C11 cDNA enzymes into EETs. CONCLUSIONS: The EETs thus formed will be released and activate K+ channels, increase outward K+ current, and hyperpolarize the plasma membrane. The resulting membrane hyperpolarization inhibits voltage-gated Ca2+ channels and leads to arteriolar dilation, thereby increasing regional nutritive blood flow in response to neuronal activity.