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 (nNOSÀ/À) mice exhibited exacerbated histological injury after middle cerebral artery occlusion (MCAO) relative to wild-type (WT) females, unlike the protection observed in male nNOSÀ/À 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 (PARP1À/À), female PARP1À/À 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, 17b 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.

The restoration of energy balance during ischemia is critical to cellular survival; however, relatively little is known concerning the regulation of neuronal metabolic pathways in response to central nervous system ischemia. AMP-activated protein kinase (AMPK), a master sensor of energy balance in peripheral tissues, is phosphorylated and activated when energy balance is low. We investigated whether AMPK might also modulate neuronal energy homeostasis during ischemia. We utilized two model systems of ischemia, middle cerebral artery occlusion in vivo and oxygen-glucose deprivation in vitro, to delineate changes in AMPK activity incurred from a metabolic stress. AMPK is highly expressed in cortical and hippocampal neurons under both normal and ischemic conditions. AMPK activity, as assessed by phosphorylation status, is increased following both middle cerebral artery occlusion and oxygen-glucose deprivation. Pharmacological inhibition of AMPK by either C75, a known modulator of neuronal ATP levels, or compound C reduced stroke damage. In contrast, activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside exacerbated damage. Mice deficient in neuronal nitric-oxide synthase demonstrated a decrease in both stroke damage and AMPK activation compared with wild type, suggesting a possible interaction between NO and AMPK activation in stroke. These data demonstrate a role for AMPK in the response of neurons during metabolic stress and suggest that in ischemia the activation of AMPK is deleterious. The ability to manipulate pharmacologically neuronal energy balance during ischemia represents an innovative approach to neuroprotection.Despite significant advances in our understanding of neuronal responses to ischemia, interventions for stroke remain elusive. A reduction in oxygen and glucose in cells causes a disruption of protein synthesis, depletion of intracellular energy stores, destabilization of the cell membrane, opening of voltagegated Ca 2ϩ channels, and activation of the N-methyl-D-aspartic acid receptor. These conditions lead to excitotoxic and oxidative damage (1). Stimulation of nitric-oxide synthase (NOS) 1 by increasing Ca 2ϩ levels causes accumulations of nitric oxide (NO), superoxide, peroxynitrite (ONOO), and free radicals, which further damage the cell membrane and may lead to DNA damage (1, 2). In the attempt to repair damage and return neurons to homeostasis, numerous energy-consuming processes are activated (3, 4). Overactivation of these pathways during ischemia can lead to complete energy failure and cell death (5, 6). The mechanisms by which neurons attempt to restore energy balance are largely unknown.In peripheral tissues, AMP-activated protein kinase (AMPK), a member of a metabolite-sensing protein kinase family (7,8), is activated by energy deficiency to coordinate a switch from anabolic to catabolic pathways to produce a positive energy balance. AMPK is composed of a catalytic ␣ subunit (␣1 or ␣2) and two regulatory subunits (␤ and ␥) (9) and is activated via phosphorylation by an up...

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Background and Purpose-5Ј 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 ␣-1 or ␣-2. Results-AMPK inhibition was neuroprotective after focal stroke. Mice deficient in AMPK ␣-2 demonstrated significantly smaller infarct volumes compared with wild-type littermates, whereas deletion of AMPK ␣-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 ␣-2 contributes to the deleterious effects of AMPK activation. AMPK inhibition leads to neuroprotection even when these agents are administered poststroke.

It is increasingly recognized that histological and functional outcomes after stroke are shaped by biologic sex. Emerging data suggests that ischemic cell death pathways are sexually dimorphic (Hurn et al., 2005; Lang and McCullough, 2008). Reducing neuronal nitric oxide (NO) or poly-ADP ribose polymerase (PARP-1) activation protects only the male brain (Hagberg et al., 2004), and paradoxically enhances ischemic injury in females (McCullough et al., 2005). In this study, we examined downstream mediators of NO/PARP activation to investigate possible mediators of ischemic sexual dimorphism. Nuclear translocation of Apoptosis Inducing Factor (AIF) was equivalent in wild-type males and females after stroke and was unaffected by estrogen exposure. Deletion of PARP1 led to a dramatic reduction in stroke-induced poly(ADP-ribose) polymerase (PAR) formation and AIF translocation in both sexes, yet ischemic damage was reduced only in males. Subsequent examination of AIF-deficient Harlequin mice demonstrated that male Harlequin mice had less PAR formation, reduced AIF translocation and less ischemic damage than male wild-type mice. In contrast, female Harlequin mice had no neuroprotective effect of gene deletion despite robust reductions in PAR formation and AIF translocation. Although equivalent activation of this cell death pathway occurs in both sexes after ischemia, detrimental effects are only present in males. AIF translocation and PAR formation do not mediate ischemic injury in the female brain, therefore agents designed to reduce PARP1 activation are unlikely to benefit females.

Stroke is a leading cause of permanent disability and death. It is well accepted that the principal mammalian estrogen (E2), 17-b estradiol, provides robust neuroprotection in a variety of brain injury models in animals of both sexes. E2 enhances neurogenesis after stroke in the subventricular zone; however, it is unknown if these cells survive long-term or enhance functional recovery. In this study, we examined stroke-induced neurogenesis in male, gonadally intact female, and ovariectomized female mice 2 and 6 weeks after stroke. Treatment with 17-b estradiol increased 5-bromo-2 0 -deoxyuridine-labeled cells at both time points in both the dentate gyrus and subventricular zone; the majority were colabeled with doublecortin at 2 weeks and with NeuN at 6 weeks. Stroke-induced neurogenesis was reduced in estrogen receptor knockout mice, as well as in mice lacking the gene for aromatase, which converts testosterone into E2. Improved behavioral deficits were seen in E2-treated mice, suggesting that E2-induced increases in poststroke neurogenesis contribute to poststroke recovery.

Female patients experience substantial neuroprotection after experimental stroke compared with male patients, a finding attributed to the protective effects of gonadal hormones. This study examined the response of male- and female-derived organotypic hippocampal slices to oxidative and excitotoxic injury. Both oxygen and glucose deprivation and N-methyl-D-aspartic acid exposure led to neuronal death; however, female-derived cultures sustained less injury than male-derived cultures. Cell death after oxygen and glucose deprivation was ameliorated in male cultures, but not female cultures, by the addition of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor. These studies have relevance to researchers investigating neuroprotective agents in mixed sex experiments.

Activation of Akt has been implicated as a major contributor to neuronal survival after an ischemic insult. Numerous neuroprotective agents have been shown to augment Akt activity, suggesting that this protein represents a major mechanism of cellular salvage after injury. Estrogen is known to augment Akt, but the possibility that Akt plays a differential role in the male and female brain has yet to be evaluated. In this study, we employed both pharmacological and genetic approaches to investigate the role of Akt in stroke. Utilizing a focal stroke model we show that deletion of the Akt1 isoform does not affect stroke outcome in either male or female mice. Akt1 deficient mice had equivalent levels of phosphorylated Akt (p-Akt) when compared to their WT controls following stroke suggesting that alternative isoforms can compensate for Akt1 loss. Secondly, estrogen's neuroprotective effect is maintained in Akt1 −/− mice and estrogen exposure did not enhance p-Akt levels in WT female mice. Thirdly, we show that inhibiting Akt using the direct pan-Akt inhibitor triciribine has no effect on stroke outcome despite dramatic reductions in p-Akt. Our study demonstrates the limitations of genetic mouse models and suggests that the importance of Akt to ischemic outcome remains unclear.