Louise D. McCullough

Guidelines for the Prevention of Stroke in Women A Statement for Healthcare Professionals From the American Heart Association/American Stroke Association

Purpose— The aim of this statement is to summarize data on stroke risk factors that are unique to and more common in women than men and to expand on the data provided in prior stroke guidelines and cardiovascular prevention guidelines for women. This guideline focuses on the risk factors unique to women, such as reproductive factors, and those that are more common in women, including migraine with aura, obesity, metabolic syndrome, and atrial fibrillation. Methods— Writing group members were nominated by the committee chair on the basis of their previous work in relevant topic areas and were approved by the American Heart Association (AHA) Stroke Council’s Scientific Statement Oversight Committee and the AHA’s Manuscript Oversight Committee. The panel reviewed relevant articles on adults using computerized searches of the medical literature through May 15, 2013. The evidence is organized within the context of the AHA framework and is classified according to the joint AHA/American College of Cardiology and supplementary AHA Stroke Council methods of classifying the level of certainty and the class and level of evidence. The document underwent extensive AHA internal peer review, Stroke Council Leadership review, and Scientific Statements Oversight Committee review before consideration and approval by the AHA Science Advisory and Coordinating Committee. Results— We provide current evidence, research gaps, and recommendations on risk of stroke related to preeclampsia, oral contraceptives, menopause, and hormone replacement, as well as those risk factors more common in women, such as obesity/metabolic syndrome, atrial fibrillation, and migraine with aura. Conclusions— To more accurately reflect the risk of stroke in women across the lifespan, as well as the clear gaps in current risk scores, we believe a female-specific stroke risk score is warranted.

Neuroprotective Function of the PGE2 EP2 Receptor in Cerebral Ischemia

The cyclooxygenases COX-1 and COX-2 catalyze the first committed step of prostaglandin synthesis from arachidonic acid. Previous studies in rodent stroke models have shown that the inducible COX-2 isoform promotes neuronal injury, and the administration of COX-2 inhibitors reduces infarct volume. We investigated the function of PGE2, a principal prostaglandin product of COX-2 enzymatic activity, in neuronal survival in cerebral ischemia. PGE2 exerts its downstream effects by signaling through a class of four distinct G-protein-coupled EP receptors (for E-prostanoid: EP1, EP2, EP3, and EP4) that have divergent effects on cAMP and phosphoinositol turnover and different anatomical distributions in brain. The EP2 receptor subtype is abundantly expressed in cerebral cortex, striatum, and hippocampus, and is positively coupled to cAMP production. In vitro studies of dispersed neurons and organotypic hippocampal cultures demonstrated that activation of the EP2 receptor was neuroprotective in paradigms of NMDA toxicity and oxygen glucose deprivation. Pharmacologic blockade of EP2 signaling by inhibition of protein kinase A activation reversed this protective effect, suggesting that EP2-mediated neuroprotection is dependent on cAMP signaling. In the middle cerebral artery occlusion-reperfusion model of transient forebrain ischemia, genetic deletion of the EP2 receptor significantly increased cerebral infarction in cerebral cortex and subcortical structures. These studies indicate that activation of the PGE2 EP2 receptor can protect against excitotoxic and anoxic injury in a cAMP-dependent manner. Taken together, these data suggest a novel mechanism of neuroprotection mediated by a dominant PGE2 receptor subtype in brain that may provide a target for therapeutic intervention.

Haloperidol and nucleus accumbens dopamine depletion suppress lever pressing for food but increase free food consumption in a novel food choice procedure

An important aspect of motivated behavior is that organisms will perform complex instrumental behaviors to gain access to stimuli such as food. In the present study, food-deprived rats were tested in an operant chamber in which the animals had a choice between pressing a lever to obtain a more-preferred food (Bioserve pellets), or free feeding on a less-preferred food (lab chow). Typically, rats pressed the lever to obtain the preferred food pellets, and ate little of the less-preferred food even though it was freely available. Pre-fed rats showed suppression of both lever pressing and feeding. Systemic administration of 0.1 mg/kg haloperidol (HP) led to a dramatic shift in the behavior of these rats, such that the number of lever presses was substantially reduced, but the amount of less-preferred food consumed showed a significant increase. This result occurred if the rats pressed a lever on either a CRF or FR5 schedule. Injection of 3.5–7.0 µg HP directly into the nucleus accumbens, or intra-accumbens injections of 6-hydroxy-dopamine, also decreased lever pressing for food and increased feeding on laboratory chow. Thus, interference with brain dopamine suppressed a highly active instrumental response for food, although the behavior of the animal was still directed towards food acquisition and consumption.

Estrogen and ischemic neuroprotection: An integrated view

Women are protected from stroke in their early years relative to men. This native neuroprotection is lost within ten years of the menopause, an observation commonly attributed to loss of estrogen with age. Data are now available from large, randomized, clinical trials that question the use of hormone replacement therapy (HRT) for either the primary or secondary prevention of vascular disease and stroke. In contrast to these studies of disease prevention, evidence from the bench suggests that estrogen is a potent neuroprotectant, demonstrating cell salvage from ischemic death pathways. This apparent dichotomy between the limitation of HRT in ameliorating complex disease development vs the excellent performance of estradiol in containing experimental brain damage remains to be understood.

Ischemic Nitric Oxide and Poly (ADP-Ribose) Polymerase-1 in Cerebral Ischemia: Male Toxicity, Female Protection

It is well established that tissue damage and functional outcome after experimental or clinical stroke are shaped by biologic sex. We investigated the novel hypothesis that ischemic cell death from neuronally derived nitric oxide (NO) or poly-ADP ribose polymerase (PARP-1) activation is sexually dimorphic and that interruption of these molecular death pathways benefits only the male brain. Female neuronal nitric oxide synthase (nNOS) knockout (nNOS−/−) mice exhibited exacerbated histological injury after middle cerebral artery occlusion (MCAO) relative to wild-type (WT) females, unlike the protection observed in male nNOS−/− littermates. Similarly, treatment with the nNOS inhibitor (7-nitroindozole, 25 mg/kg) increased infarction in female C57Bl6 WT mice, but protected male mice. The mechanism for this sexually specific response is not mediated through changes in protein expression of endothelial NOS or inducible NOS, or differences in intraischemic cerebral blood flow. Unlike male PARP-1 knockouts (PARP1−/−), female PARP1−/− littermates sustained grossly increased ischemic damage relative to sex-matched WT mice. Treatment with a PARP inhibitor (PJ-34, 10 mg/kg) resulted in identical results. Loss of PARP-1 resulted in reversal of the neuroprotective activity by the female sex steroid, 17β estradiol. These data suggest that the previously described cell death pathways involving NO and PARP ischemic neurotoxicity may be operant solely in male brain and that the integrity of nNO/PARP-1 signaling is paradoxically protective in the female.

Chronic behavioral testing after focal ischemia in the mouse: functional recovery and the effects of gender

Several useful behavioral tests exist for measuring behavioral recovery after ischemia in higher-order animals and rats. With the increasing use of mice in focal stroke research, simple, reliable, and reproducible behavioral testing has become a priority. As neuroprotective agents are tested, long-term outcome must be assessed, especially in studies focused on neuronal plasticity and regeneration after ischemia. Our laboratory and others have previously shown that estrogen (E2) is neuroprotective in rodent stroke paradigms. We examined a battery of behavioral tests in male and female mice subjected to 90 min of middle cerebral artery occlusion (MCAO) to determine the most sensitive tests for detecting sensorimotor dysfunction after stroke, and to determine the functional significance of E2-mediated neuroprotection. Only two tests, the corner test and the cylinder test, were able to differentiate between groups (sham and stroke) after several days of repeated testing. The cylinder test was sensitive to the neuroprotective/neurorestorative effects of E2, but 2 weeks after stroke, the cylinder test was unable to distinguish between sham and stroke animals treated with E2. In contrast, the corner test was able to differentiate stroke and sham animals even 6 weeks after stroke, but did not distinguish animals treated with E2 vs. vehicle. These tests provide a simple, rapid, reliable assessment of sensorimotor dysfunction in the mouse after focal ischemia. Hormonal status influences speed of recovery on cylinder testing in animals of both genders. This suggests that a short battery of tests including the neurological score, cylinder, and corner test may be adequate to rapidly and repeatedly assess sensorimotor dysfunction in mice of both genders.

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 mm3; 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.

Aromatase Cytochrome P450 and Extragonadal Estrogen Play a Role in Ischemic Neuroprotection

Female animals are protected from many forms of neurological injury and degeneration relative to their male counterparts, in part attributable to their native estrogens. We hypothesized that estradiol aromatized from precursor androgens via the cytochrome P450 aromatase contributes to ischemic neuroprotection in the female. Female homozygous aromatase knock-out (ArKO) mice and randomly cycling, wild-type (WT) female littermates were treated with reversible middle cerebral artery occlusion (90 min; 22 hr reperfusion). Total and regional ischemic damage was greater in female ArKOs (total, 33.5 ± 4.8%; cortical, 47.4 ± 5.7%; striatal, 44.8 ± 7.8%) compared with WT (total, 14.2 ± 5%; cortical, 14.2 ± 4.5%; striatal, 17.5 ± 8%). Baseline blood pressure and intra-ischemic cortical perfusion were comparable in knock-outs and WT, suggesting that vascular factors do not explain ArKO ischemic sensitivity. Injury was smaller in ovariectomized WT than in ArKO, emphasizing that extragonadal local estradiol plays a critical role in females. Similar increases in cortical and striatal damage were observed in female WT mice chronically treated with the aromatase inhibitor fadrozole compared with vehicle-treated control mice. Restoration of plasma 17β-estradiol to physiological levels completely reversed the ArKO females susceptibility to injury. These findings indicate that the biosynthetic enzyme P450 aromatase is key to endogenous neuroprotection in females and suggest that enhancing local, nongonadal estrogen formation could have therapeutic implications is ischemic neuropathology.

Neuroprotective Effects of Adenosine Monophosphate-Activated Protein Kinase Inhibition and Gene Deletion in Stroke

Background and Purpose— 5′ adenosine monophosphate-dependent protein kinase (AMPK) acts as a metabolic sensor. AMPK is elevated under ischemic conditions, but the role of AMPK in ischemic brain remains controversial. In this study, we examined the effects of AMPK inhibition using both pharmacological and genetic approaches in an in vivo stroke model. Methods— Focal stroke was induced by reversible middle cerebral artery occlusion in male wild-type mice as well as mice deficient in one of the isoforms of the catalytic subunit of AMPK, AMPK α-1 or α-2. Results— AMPK inhibition was neuroprotective after focal stroke. Mice deficient in AMPK α-2 demonstrated significantly smaller infarct volumes compared with wild-type littermates, whereas deletion of AMPK α-1 had no effect. Phosphorylation of a major upstream regulator of AMPK, LKB1, was also induced in stroke brain. Conclusions— AMPK activation is detrimental in a model of focal stroke. The AMPK catalytic isoform α-2 contributes to the deleterious effects of AMPK activation. AMPK inhibition leads to neuroprotection even when these agents are administered poststroke.

TTC, fluoro-Jade B and NeuN staining confirm evolving phases of infarction induced by middle cerebral artery occlusion.

Considerable debate exists in the literature on how best to measure infarct damage and at what point after middle cerebral artery occlusion (MCAO) infarct is histologically complete. As many researchers are focusing on more chronic endpoints in neuroprotection studies it is important to evaluate histological damage at later time points to ensure that standard methods of tissue injury measurement are accurate. To compare tissue viability at both acute and sub-acute time points, we used 2,3,5-triphenyltetrazolium chloride (TTC), Fluoro-Jade B, and NeuN staining to examine the evolving phases of infarction induced by a 90-min MCAO in mice. Stroke outcomes were examined at 1.5h, 6h, 12h, 24h, 3d, and 7d after MCAO. There was a time-dependent increase in infarct volume from 1.5h to 24h in the cortex, followed by a plateau from 24h to 7d after stroke. Striatal infarcts were complete by 12h. Fluoro-Jade B staining peaked at 24h and was minimal by 7d. Our results indicated that histological damage as measured by TTC and Fluoro-Jade B reaches its peak by 24h after stroke in a reperfusion model of MCAO in mice. TTC staining can be accurately performed as late as 7d after stroke. Neurological deficits do not correlate with the structural lesion but rather transient impairment of function. As the infarct is complete by 24h and even earlier in the striatum, even the most efficacious neuroprotective therapies are unlikely to show any efficacy if given after this point.