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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Brain Res. Author manuscript; available in PMC Mar 19, 2011.
Published in final edited form as:
PMCID: PMC2836484
NIHMSID: NIHMS180559

Acute estradiol protects CA1 neurons from ischemia-induced apoptotic cell death via the PI3K/Akt pathway

Abstract

Global ischemia arising during cardiac arrest or cardiac surgery causes highly selective, delayed death of hippocampal CA1 neurons. Exogenous estradiol ameliorates global ischemia-induced neuronal death and cognitive impairment in male and female rodents. However, the molecular mechanisms by which a single acute injection of estradiol administered after the ischemic event intervenes in global ischemia-induced apoptotic cell death are unclear. Here we show that acute estradiol acts via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling cascade to protect CA1 neurons in ovariectomized female rats. We demonstrate that global ischemia promotes early activation of glycogen synthase kinase-3β (GSK3β) and forkhead transcription factor of the O class (FOXO)3A, known Akt targets that are related to cell survival, and activation of caspase-3. Estradiol prevents ischemia-induced dephosphorylation and activation of GSK3β and FOXO3A, and the caspase death cascade. These findings support a model whereby estradiol acts by activation of PI3K/Akt signaling to promote neuronal survival in the face of global ischemia.

Keywords: Estradiol, PI3K/Akt/signaling, Neuronal death, Apoptosis, Global ischemia

1. Introduction

Transient global brain ischemia arising during cardiac arrest, cardiac surgery or induced experimentally in animals via bilateral carotid artery occlusion, causes highly selective, delayed neuronal death and delayed neurological deficits (Graham and Chen, 2001; Liou et al., 2003; Lo et al., 2003; Zukin et al., 2004). Pyramidal neurons in the hippocampal CA1 are particularly vulnerable, whereas interneurons in this cell layer and pyramidal neurons in other hippocampal subfields survive. Histological evidence of CA1 pyramidal neuron degeneration is not observed until 2-3 days after global ischemia in rats (Graham and Chen, 2001; Liou et al., 2003; Lo et al., 2003; Zukin et al., 2004). Although the mechanisms underlying ischemia-induced death are as yet unclear, the substantial delay between insult and onset of death provides the opportunity to examine molecular events that destine these neurons to die.

Estradiol-17β, the primary estrogen secreted by the ovaries, acts on neurons to increase spine density and synapse number (Gould et al., 1990; Hao et al., 2003; Pozzo-Miller et al., 1999; Prange-Kiel and Rune, 2006; Sato et al., 2007; Woolley and McEwen, 1992; Woolley and McEwen, 1994), synaptic connectivity sustained by N-methyl-d-aspartate (NMDA) receptor activation (Srivastava et al., 2008), NMDA receptor NR1 subunit expression (Gazzaley et al., 1996), NR2B subunit mRNA and the number of NR2B binding sites (Cyr et al., 2001), the magnitude of long-term potentiation (LTP) (Cordoba Montoya and Carrer, 1997; Gupta et al., 2001; Smith and McMahon, 2005; Smith and McMahon, 2006) and potentiates kainate-elicited currents in CA1 pyramidal neurons (Gu and Moss, 1998; Moss and Gu, 1999). Furthermore, estrogens afford neuroprotection of cortical neurons in primary culture against chemically-induced neuronal death (Harms et al., 2001; Honda et al., 2000), in rat hippocampal organotypic cultures (Cimarosti et al., 2005b; Cimarosti et al., 2005a) and in experimental models of global and focal ischemia (Amantea et al., 2005; Behl 2002; Garcia-Segura et al., 2001; Wise, 2003) and ameliorate the cognitive deficits associated with ischemic cell death (Gulinello et al., 2006; Plamondon et al., 2006; Sandstrom and Rowan, 2007; Soderstrom et al., 2009). Estrogen receptor (ER)-α and ER-β are expressed in the hippocampus where they could subserve the neuroprotective actions of estradiol (McEwen, 2002; Miller et al., 2005). Moreover, neuroprotection by estradiol may involve interactions with membrane-associated estrogen receptors, together with intracellular ERs, to activate cell signaling pathways that promote neuronal survival (Lebesgue et al., 2009).

Crosstalk between estradiol and growth factor signaling pathways is implicated in the cellular actions of estradiol. In the brain, estradiol activates extra-cellular-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) (Cardona-Gomez et al., 2002a) and phosphoinositide 3-kinase (PI3K) (Segars and Driggers, 2002), well-characterized intracellular signaling cascades implicated in neuronal plasticity and survival (Alessi et al., 1996; Behl, 2002; Bryant et al., 2006; Datta et al., 1999; Sweatt, 2004; Thomas and Huganir, 2004). Chronic estradiol at a physiological dose acts via classical ER-α and ER-β, insulin-like growth factor-1 receptors, ERK/MAPK and cAMP response element binding protein (CREB) signaling to promote neuronal survival after transient global ischemia (Jover-Mengual et al., 2007; Miller et al., 2005). A single injection of 17β-estradiol administered to ovariectomized rats 2-4 days before ischemia also protects hippocampal neurons against ischemic damage via activation of CREB (Raval et al., 2009). Moreover, a single dose of estradiol administered immediately after reperfusion (acute estradiol) ameliorates global ischemia-induced neuronal death and cognitive deficits (Gulinello et al., 2006), but the mechanism of this protection has not been explored.

Treatment of rat hippocampal organotypic cultures with estradiol induce the phosphorylation of the serine-threonine protein kinase B (Akt), an effector immediately downstream of PI3K (Cimarosti et al., 2005b) and a key player in the apoptotic neuronal death machinery after focal (Noshita et al., 2001; Noshita et al., 2003) and global (Endo et al., 2006; Miyawaki et al., 2009) cerebral ischemia. Several targets of Akt are involved in its ability to foster cell survival. Akt promotes cell survival, at least in part, by phosphorylation and inactivation of proapoptotic downstream targets such as glycogen synthase kinase-3β (GSK3β) (Cross et al., 1995; Endo et al., 2006), the pro-apoptotic forkhead transcription factor family member, forkhead transcription factor of the O class (FOXO)3A (Brunet et al., 2001; Kawano et al., 2002) and Bad (Datta et al., 1997; Miyawaki et al., 2008; Saito et al., 2003). Akt also controls a critical prosurvival protein, β-catenin, by modulating the activity of GSK3β. GSK3β can promote cell injury (Crowder and Freeman, 2000) and increase caspase-3 activity (Koh et al., 2003), and these actions are reduced when Akt phosphorylates and inactivates GSK3β (Nishimoto et al., 2008). There is evidence that estradiol acts via Akt to maintain FOXO3A phosphorylation and activation in the face of focal ischemia (Won et al., 2006).

The present study was undertaken to identify intracellular signaling cascades that mediate acute estradiol neuroprotection in global ischemia. We show that estradiol acts via PI3K/Akt signaling to promote survival of hippocampal CA1 pyramidal neurons after transient global ischemia. Global ischemia promotes a transient increase of Akt phosphorylation and decrease in the phosphorylation of Akt targets GSK3β and FOXO3A in the hippocampal CA1 in the first few hours after ischemia. Estradiol prevents ischemia-induced dephosphorylation and activation of GSK3β and FOXO3A and caspase-3 activation. Thus, estradiol administered acutely after ischemia maintains PI3K/Akt signaling, thereby promoting neuronal survival in the face of global ischemia.

2. Results

2.1. PI3K/Akt signaling is critical to estradiol protection of CA1 neurons

Estradiol acts via PI3K to afford protection of cortical neurons in primary culture (Harms et al., 2001; Honda et al., 2000; Honda et al., 2001) and in rat organotypically cultured hippocampal slices against chemically-induced neuronal death (Cimarosti et al., 2005b). We first examined a possible role for PI3K/Akt signaling in estradiol protection. Ovariectomized female rats were subjected to global ischemia or sham operation and immediately infused icv with estradiol (50 μg) in vehicle or vehicle alone. Animals also received icv infusion of the PI3K inhibitor LY294002 (30 μg) or vehicle into the lateral ventricle at 0 and 12 h after surgery. Global ischemia induced extensive death of pyramidal cells in the hippocampal CA1 at 7 days post-ischemia (P < 0.001 vs. sham; compare Fig. 1i,m,q with Fig. 1a,e,q). Estradiol did not detectably alter the appearance or number of CA1 neurons in sham-operated rats (Fig. 1c,g,q), but greatly reduced the ischemia-induced neuronal loss (P < 0.01 vs. ischemia, Fig. 1,k,o,q). Plasma estradiol levels at 1 h after estradiol injection were 26.9 ± 3.0 pg/ml in the placebo group and 7895 ± 552 pg/ml in the estradiol group. The PI3K inhibitor LY294002 did not detectably alter the number or appearance of surviving neurons in sham-operated rats (Fig. 1b,f,d,h,q) or vehicle-treated animals subjected to ischemia (Fig. 1j,n,q), but abrogated the neuroprotective action of estradiol in the hippocampal CA1 (P < 0.001 vs. estradiol alone, Fig. 1l,p,q). These findings indicate that whereas LY294002 reverses the estradiol neuroprotection, it is itself neither toxic nor protective in the global ischemia model. Together, these findings indicate that PI3K/Akt signaling is critical to estradiol protection of hippocampal neurons in a rat model of global ischemia.

Fig. 1
The PI3K inhibitor LY294002 attenuates estradiol protection

2.2. The PI3K inhibitor LY294002 attenuates global ischemia- induced increase in Akt phosphorylation in CA1

To examine the effects of the PI3K inhibitor LY294002 on the abundance and phosphorylation status of Akt, ovariectomized rats were subjected to global ischemia or sham operation, treated with the PI3K inhibitor LY294002 (30 μg) or vehicle and examined for Akt and p-Akt abundance in CA1 after reperfusion. Global ischemia markedly increased phosphorylation of Akt at Ser473 in the CA1 pyramidal cell layer (increase to 354% of sham-operated, vehicle-infused control; P < 0.01; Fig. 2a,b). LY LY294002 did not affect Akt phosphorylation in sham-operated animals but significantly reversed the effects of ischemia on p-Akt in CA1 (Fig. 2a,b). These findings indicate that the dose of LY294002 used effectively decreased Akt signaling in the hippocampus after ischemia.

Fig. 2
The PI3K inhibitor LY294002 attenuates global ischemia- induced increase in Akt phosphorylation in CA1

2.3. Estradiol increases Akt phosphorylation in CA1 neurons

To examine the effects of ischemia and estradiol on the abundance and phosphorylation status of Akt, ovariectomized rats were subjected to global ischemia or sham operation, treated with estradiol or vehicle and examined for Akt and p-Akt abundance in CA1 at 1, 3 and 24 h after reperfusion. Global ischemia significantly increased phosphorylation of Akt at Ser473 in the CA1 pyramidal cell layer, evident at 1 h after ischemia (increase to 193% of control (sham-operated, vehicle-infused); P < 0.001; Fig. 3a,b); at 3 and 24 h, p-Akt levels were not significantly different from controls (Fig. 3a,b). Estradiol significantly increased Akt phosphorylation in sham-operated animals at 1 h (increase to 139% of control, P < 0.05, Fig. 3a,b) but did not significantly alter Akt phosphorylation at times after global ischemia (Fig. 3a,b).

Fig. 3
Estradiol and global ischemia transiently increase Akt phosphorylation in CA1

2.4. Estradiol prevents dephosphorylation and inactivation of ERK2 in postischemic CA1

Estradiol is an upstream regulator of ERK/MAPK signaling in hippocampal neurons (Bi et al., 2000; Cardona-Gomez et al., 2002b), and ERK/MAPK is critical to the ability of long-term estradiol pretreatment to protect hippocampal neurons after global ischemia (Jover-Mengual et al., 2007). To compare the effects of post-ischemic administration of estradiol with our previous work implicating this signaling pathway in estradiol’s neuroprotective actions when hormone is provided chronically at low levels, we examined the status of ERK1/2 phosphorylation after acute estradiol administration. Ovariectomized rats were subjected to global ischemia or sham operation, treated with estradiol or vehicle, and protein samples from the CA1 were subjected to Western blot analysis and examined for ERK1/2 abundance and phosphorylation at 1 and 3 h after reperfusion. Global ischemia significantly reduced phosphorylation of ERK1 and ERK2 in CA1, evident at 1 h after ischemia (decrease by ~36% for p-ERK1, P < 0.05 vs. sham-operated animals; decrease by ~51% for p-ERK2, P < 0.001 vs. sham-operated animals, Fig. 4a,b,c); at 3 h, p-ERK1/2 levels were not significantly different from controls (Fig. 4a,b,c). Estradiol did not significantly alter ERK1 and ERK2 phosphorylation in sham-operated animals but prevented the early ischemia-induced dephosphorylation of ERK2 (P < 0.01 vs. ischemia at 1 h, Fig. 4a,c). In estradiol-treated animals, ischemia did not reduce phosphorylation of ERK1 at 1 h after reperfusion (Fig. 4a,b).

Fig. 4
Estradiol prevents ischemia-induced dephosphorylation of ERK2 in CA1

2.5. Estradiol increases GSK-3β phosphorylation 3 h after ischemia in CA1 neurons

GSK-3β is a non-receptor serine/threonine kinase and a downstream target of Akt implicated in estradiol neuroprotection (Mendez and Garcia-Segura, 2006). Akt phosphorylates GSK-3β on serine 9 to render it inactive, thereby activating glycogen synthesis and preventing apoptosis. To examine the effects of estradiol treatment and ischemia on GSK-3β abundance and phosphorylation status, rats were subjected to global ischemia or sham operation, administered a single, acute injection of estradiol or vehicle, and protein samples from the CA1 were subjected to Western blot analysis at 1 and 3 h after reperfusion. Global ischemia did not significantly change the levels of p-GSK3β at any times examined (Fig. 5a,b). Estradiol significantly increased GSK-3β phosphorylation at 3 h after ischemia (P < 0.05 vs. ischemia at 3 h, vehicle-treated, Fig. 5a,b).

Fig. 5
Estradiol prevents ischemia-induced inhibition of GSK-3β phosphorylation in hippocampal CA1

2.6. Estradiol prevents ischemia-induced dephosphorylation and activation of FOXO3A in CA

A well-characterized downstream target of PI3K/Akt signaling is the transcription factor FOXO3A, which promotes transcription of genes implicated in death pathways (Brunet et al., 2001; Kawano et al., 2002; Miyawaki et al., 2008; Miyawaki et al., 2009). Akt directly phosphorylates FOXO family members and inhibits their ability to induce expression of death genes. Akt-induced phosphorylation of FOXO3A retains the molecule in the cytoplasm, away from target genes in the nucleus. To examine whether estradiol regulates phosphorylation and inactivation of FOXO, ovariectomized rats were subjected to global ischemia or sham operation, treated with estradiol or vehicle and examined for FOXO3A and p-FOXO3A abundance in CA1 at 3 h after reperfusion. Global ischemia induced a significant decrease in p-FOXO3A (by ~37%, P < 0.05 vs. sham-operated animals, Fig. 6a,b), with no significant change in total FOXO3A abundance in the cytosolic fraction of CA1. Estradiol significantly increased FOXO3A phosphorylation in sham-operated animals (increase to 142% of control, P < 0.05, Fig. 6a,b) and prevented the ischemia-induced dephosphorylation and activation of FOXO3A at 3 h after ischemia in the vulnerable CA1 (P < 0.01 vs. vehicle-treated, Fig. 6a,b).

Fig. 6
Estradiol prevents ischemia-induced FOXO3A dephosphorylation

2.7. Estradiol blocks ischemia-induced activation of caspase-3 activity in CA1 neurons

Injurious stimuli such as global ischemia disrupt the integrity of the mitochondrial membrane, leading to the release of cytochrome c and activation of caspase-3, a “terminator” caspase implicated in the execution step of apoptosis (for review, see (Bredesen, 2008; Zukin et al., 2004)). Global ischemia promotes cleavage of the biologically inactive precursor procaspase-3 to generate activated caspase-3 (Jover et al., 2002); ischemia-induced caspase-3 activity is maximal at 24 h after insult (Tanaka et al., 2004). To directly measure caspase-3-like functional activity after ischemia, we labeled brain sections with FAM-DEVD-FMK, a fluorescein-tagged analogue of the caspase inhibitor zDEVD-FMK, at 24 h. FAM-DEVD-FMK enters cells and binds irreversibly to catalytically active caspase-3, and thus provides a fluorescent indicator of the abundance of active caspase-3. In brain sections from control (sham-operated, vehicle-infused) animals, caspase activity was low (Fig. 7a,b, g). Global ischemia induced a 16-fold increase in caspase activity in the hippocampal CA1, evident at 24 h (P < 0.05 vs. control; Fig. 7c,d,g). The increase in caspase activity was subfield-specific in that it was not observed in the resistant CA3 or dentate gyrus. Acute estradiol treatment blocked the ischemia-induced elevation of caspase-3 activity in CA1 (P < 0.05 vs. vehicle-treated, Fig. 7e,f,g).

Fig. 7
Estradiol blocks ischemia-induced caspase-3-like activity in CA1

3. Discussion

These findings provide clear evidence implicating the Akt pathway as a critical cellular mediator of the neuroprotection afforded by a supraphysiological dose of estradiol administered at the onset of reperfusion in a clinically relevant model of global ischemia. We now have evidence that icv administration of a much lower dose (2 μg) is just as effective as the high dose (unpublished observations) and that LY 294002 also blocks protection by the low dose. These results are in agreement with findings of others that Akt is critical to cell survival after cerebral ischemia and indicate that hormone administration after an ischemic event can maintain Akt signaling. Activation of Akt and suppression of GSK3β mediates neuroprotection of vulnerable hippocampal CA1 neurons after transient global ischemia by overexpression of copper/zinc-superoxide dismutase (Endo et al., 2006) or by ischemic preconditioning (Miyawaki et al., 2008; Yano et al., 2001). Estradiol acts via PI3K to afford protection of cultured cortical neurons subjected to chemically-induced death (Harms et al., 2001; Honda et al., 2000) and of neurons in organotypically cultured hippocampal slices subjected to oxygen-glucose deprivation (Cimarosti et al., 2005b). PI3K/Akt signaling participates in the neuroprotective actions of estradiol pretreatment in gerbils subjected to focal ischemia (Koh et al., 2006). We now document the involvement of Akt in the neuroprotection afforded by a single, acute injection of estradiol delivered at the time of reperfusion in a clinically relevant model of global ischemia in rats.

PI3K/Akt and its downstream targets in acute estradiol neuroprotection

Our findings are consistent with the hypothesis that a high dose of estradiol administered immediately after induction of global ischemia acts via PI3K/Akt signaling to promote survival of post-ischemic neurons. Administration of the PI3K inhibitor LY294002 blocks the ability of estradiol to promote survival of CA1 pyramidal neurons in the post-ischemic hippocampus. The finding that LY294002 inhibits Akt phosphorylation in CA1 after global ischemia and blocks estradiol protection documents a role for PI3K signaling in preservation of ischemic hippocampal neurons and is consistent with studies in organotypic cultures of rat hippocampus subjected to oxygen and glucose deprivation (Cimarosti et al., 2005b). Ischemia promotes a transient increase of Akt phosphorylation in the hippocampal CA1, while phosphorylation of GSK3β and FOXO3A (known Akt targets) decrease in the first few hours after ischemia, in confirmation of others (Endo et al., 2006; Namura et al., 2000; Yano et al., 2001). At later times, activation of caspase-3 is also evident.

It is notable that Akt phosphorylation is markedly enhanced, but p-Akt is not catalytically active, in post-ischemic hippocampal neurons. Global ischemia triggers hyperphosphorylation and activation of Akt, which in turn promotes induction of the endogenous inhibitor of Akt, carboxyl-terminal modulator protein (CTMP); upon induction, CTMP rapidly binds Akt and extinguishes Akt activity (Miyawaki et al., 2009). A possible scenario is that estradiol suppresses expression of CTMP (or other negative regulators of Akt), enabling p-Akt to be activated in postischemic CA1 and promote phosphorylation and inactivation of downstream targets of Akt implicated in the apoptotic cell death, such as GSK-3β and FOXO3A.

Estradiol administered icv immediately after reperfusion prevents ischemia-induced dephosphorylation and activation of GSK3β and FOXO3A as well as caspase-3 activation. These findings are consistent with the evidence that binding of estradiol to ER-α leads to formation of a macromolecular signaling complex that recruits downstream signaling molecules such as the regulatory subunit of PI3K (Kahlert et al., 2000; Mendez et al., 2003). However, this study did not identify the cellular mediator of estradiol action when given acutely. Estradiol can activate both ER-α and ER-β as they have similar (nM) affinity for estradiol. Moreover, we have implicated both receptors in the neuroprotective actions of estradiol when administered systemically for 2 weeks prior to global ischemia (see Miller et al., 2005).

Neuroprotective pretreatments such as estradiol and ischemic preconditioning can reduce global ischemia-induced cell death by activation of Akt and subsequent inactivation of its downstream target, the proapoptotic protein Bad (Koh et al., 2006; Miyawaki et al., 2008). Our results extend these findings by demonstrating that acute estradiol also regulates two other downstream targets of Akt implicated in the apoptotic cell death, GSK-3β and FOXO3A. These molecules as well as another Akt target, mTOR, have been implicated in estradiol protection in a focal ischemia model (Won et al., 2006; Koh et al., 2006; Koh et al., 2008). Taken together, these observations support a model whereby estradiol administered acutely after insult acts via PI3K/Akt and downstream signaling molecules to promote neuronal survival in the face of ischemic insults.

Estradiol/ERK/MAPK interactions in estradiol neuroprotection

In addition to acting through the PI3K/Akt pathway, estradiol is known to activate MAPK signaling in CA1 neurons. Long-term pretreatment with estradiol at physiological levels ameliorates global ischemia-induced CA1 neuronal death (Gulinello et al., 2006; Jover et al., 2002; Miller et al., 2005). ERK/MAPK signaling is critical to estradiol-induced phosphorylation and activation of CREB and protection of CA1 neurons in global ischemia (Jover-Mengual et al., 2007). Chronic estradiol increases basal phosphorylation of both ERK1 and ERK2 in hippocampal CA1 and prevents ischemia-induced dephosphorylation and inactivation of ERK1 and CREB, downregulation of Bcl-2 and activation of the caspase death cascade. In the present study, we examined the impact of a single, acute injection of estradiol given immediately after ischemia on ERK1/2 phosphorylation/activation. Acute estradiol prevented ischemia-induced dephosphorylation of ERK2 in the early postischemic period. These findings suggest that estradiol can activate multiple signaling pathways, depending on the dose and mode of administration, which may converge on common downstream signaling molecules to promote survival of hippocampal neurons in response to transient global ischemia. Whether ERK/MAPK signaling interacts with the PI3/Akt pathway at some point or if they independently converge on a downstream target such as caspase is currently unknown.

In summary, our results indicate that the neuroprotective actions of estradiol administered at the onset of reperfusion in a clinically relevant model of transient global ischemia are mediated by PI3K/Akt signaling, which prevents ischemia-induced activation of GSK3β and FOXO3A and the caspase death cascade. Thus, post-ischemia estrogen therapy may represent a viable strategy for rescue of neurons from global ischemia-induced cell death.

4. Experimental procedures

4.1. Animals

Age-matched female Sprague-Dawley rats weighing 100-150 g (Charles River, Wilmington, DE) at the time of ischemic insult were maintained in a temperature and light-controlled environment with a 14 h light/10 h dark cycle and were treated in accordance with the principles and procedures of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Protocols were approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine. All female rats were ovariohysterectomized under halothane anesthesia (4% for induction, followed by 1% for maintenance).

4.2. Global ischemia

Seven days after the ovariohysterectomy, rats were subjected to global ischemia by four-vessel occlusion as described (Calderone et al., 2003). In brief, rats were fasted overnight and anesthetized with halothane (4% for induction, followed by 1% for maintenance). The vertebral arteries were subjected to electrocauterization, the common carotid arteries were exposed and isolated with a 3-0 silk thread, and the wound was sutured. Twenty-four hours later, the animals were anesthetized again, the wound was reopened and both carotid arteries were occluded for 10 min with nontraumatic aneurism clips, followed by reperfusion. Arteries were visually inspected to ensure adequate reflow. Sham-operated rats were subjected to the same anesthesia and surgical procedures as animals subjected to global ischemia (vertebral artery coagulation and carotid artery exposure), except that the carotid arteries were not occluded.

In all cases, anesthesia was discontinued immediately after initiation of carotid artery occlusion. The anesthesia was initiated again just after the non-traumatic aneurism clips were removed and maintained until the icv injections were complete (see below). In total, animals were under anesthesia 5 min before carotid artery occlusion and again for about 15 min beginning just after reperfusion to inject drugs. Body temperature was monitored and maintained at 37.5 ± 0.5 °C with a rectal thermistor and heat lamp until recovery from anesthesia. Animals that failed to show complete loss of the righting reflex and dilation of the pupils from 2 min after occlusion was initiated until the end of occlusion and the rare animals that exhibited obvious behavioral manifestations (abnormal vocalization when handled, generalized convulsions, hypoactivity) or loss of > 20% body weight by 3-7 days after ischemia were excluded from the study. Ninety three rats were subjected to global ischemia. There were 3 deaths due to respiratory arrest; 9 other rats were excluded from the study because they failed to show neurological signs of ischemia (no loss of consciousness or incomplete dilation of the pupils during occlusion).

4.3. Estradiol and LY294002 administration

Halothane-anesthetized animals were injected with 50 μg of estradiol (cyclodextrin-encapsulated 17β-estradiol, water soluble, Sigma; Saint Louis, MO) or vehicle (2-hydroxypropyl-β-cyclodextrin, 1.08 mg, Sigma) in 5 μl of saline by unilateral injection into the right lateral ventricle at a flow rate of 5 μl/min immediately after reperfusion. Some animals were injected with the PI3K inhibitor LY294002 (30 μg, Promega; Madison, WI) or vehicle (5 μl of 50% DMSO) immediately after estradiol or vehicle injection and again 12 h later. Intracerebroventricular (icv) injections of 75% DMSO have no obvious harmful effects (Blevins et al., 2002; Jover-Mengual et al., 2007). Animals were positioned in a Kopf small animal stereotaxic frame with the incisor bar lowered 3.3 ± 0.4 mm below horizontal zero. A stainless steel cannula (28 gauge) was lowered stereotaxically into the right lateral ventricle to a position defined by the following coordinates: 0.92 mm posterior to bregma, 1.2 mm lateral to bregma, 3.6 mm below the skull surface according to the atlas of Paxinos and Watson (Paxinos 1998).

4.4. Histological analysis

Neuronal cell loss was assessed by histological examination of toluidine blue-stained brain sections at the level of the dorsal hippocampus from vehicle- and estradiol-infused animals killed at 7 days after ischemia. Animals were deeply anesthetized with pentobarbital (50 mg/kg, ip), blood was collected by cardiac puncture for assay of plasma estradiol levels (see below) and perfused transcardially with ice cold 4% paraformaldehyde in PBS (0.1 M, pH 7.4). Brains were removed and immersed in fixative (4°C overnight). Coronal sections (15 μm) were cut at the level of the dorsal hippocampus (3.3 to 4.0 mm posterior from bregma) with an electronic cryostat (Thermo Electron Corporation, Pittsburgh, PA), and every fourth section was collected and stained with toluidine blue. The number of surviving pyramidal neurons per 250-μm length of the medial CA1 pyramidal cell layer was counted bilaterally in 4 sections per animal as described (Jover-Mengual et al., 2007; Miller et al., 2005) under a light microscope at 40× magnification. Cell counts from the right and left hippocampus on each of the four sections were averaged to provide a single value (number of neurons/250 μm length) for each animal.

4.5. Serum estradiol assay

Tubes containing whole blood were placed on ice (10 min) and centrifuged at 300 × g for 5 min. Serum was collected and stored (−20°C) until analyzed. Serum hormone levels were measured by fluoroimmunoassay using the DELPHIA estradiol assay (Perkin Elmer Life Sciences; Turku, Finland). All assays were performed in duplicate, and the mean value reported. The sensitivity of detection is 13 pg/ml. The inter- and intra-assay coefficients of variance are 10.1% and 4.1%, respectively.

4.6. Western blot analysis

For quantification of protein abundance in the hippocampal CA1, Western blot analysis was performed as described (Jover-Mengual et al., 2007). In brief, experimental and sham animals were deeply anesthetized with pentobarbital (50 mg/kg, ip), blood was collected by cardiac puncture for assay of plasma estradiol levels and killed by decapitation at 1, 3 and 24 h after reperfusion. Hippocampi were rapidly dissected, and transverse slices of dorsal hippocampus (1 mm) were cut with a Mcllwain tissue chopper. The CA1 was rapidly micro-dissected, placed in ice-cold saline supplemented with protease inhibitor cocktail (1%, Sigma) and phosphatase inhibitor cocktail 1 (1%, Sigma) and homogenized in lysis buffer containing HEPES (5 mM), MgCl2 (1 mM), EGTA (2 mM), dithiothreitol (1 mM), sucrose (10%), protease inhibitor cocktail (1%) and posphatase inhibitor cocktail 1 (1%). Part of the sample from each animal was used to isolate cytosolic fraction by differential centrifugation.

Proteins from whole-cell lysates (p-Akt, Akt, p-ERK1/2, ERK1/2) and cytosolic fractions (p-GSK-3β, GSK-3β, p-FOXO3A, FOXO3A) were separated by SDS-PAGE and subjected to Western blot analysis. Protein concentration was determined by BCA protein assay kit (Pierce, Rockford, IL). Aliquots of protein (40-70 μg) were dissolved in Laemmli sample buffer (0.025 M Tris-HCI, 5% glycerol, 1% SDS, 0.5% PBS, 0.1 M dithiothreitol, 2.5 mM β-mercaptoethanol, 1 mM PMSF, 0.5 mM NaHNO3, pH 6.8), loaded on 10% polyacrylamide gels, subjected to electrophoresis and transferred to nitrocellulose membranes for immunolabeling with antibodies to p-Akt, Akt, p-GSK-3β, GSK-3β, p-FOXO3A, FOXO3A, p-ERK1/2 and ERK1/2. After incubation with primary and appropriate secondary antibodies, membranes were treated with enhanced chemiluminescence reagents (ECL, Amersham Life Science) and apposed to XAR-5 X-ray film (Eastman Kodak Co., Rochester, NY). Membranes were reprobed with anti-β-actin antibody as a loading control.

To quantitate protein abundance, bands on Western blots were analyzed with a Scan Jet 4-C computing densitometer using NIH IMAGE 1.61 software. Band densities for p-Akt, p-GSK-3β, p-FOXO3A, p-ERK1 and p-ERK2 were corrected for variations in loading and normalized to the corresponding band densities for total Akt, GSK-3β, FOXO3A or total ERK1 and ERK2, respectively; normalized means were expressed as a percentage of the corresponding value for control (sham-operated, vehicle-infused) animals. Because of the large number of treatment groups, which included two surgical conditions (sham vs. ischemia), two hormone treatments (estradiol vs. vehicle) and multiple time points after surgery, it was not always possible to run samples for all conditions on a single gel. Therefore, to enable comparisons from experiment to experiment, band densities for all samples on a given gel were normalized to the band density for a sample from an animal treated with vehicle, subjected to sham operation and killed 1 h after surgery (“control”). Each gel included at least one sample from such a control animal to enable comparisons of data across different experiments, and a different control animal was prepared for each experiment.

4.7. Antibodies

The following antibodies were used in this study: 1) anti-phospho Akt rabbit polyclonal antibody, which recognizes Akt only when phosphorylated at Ser473 (1:1000; Cell Signaling Technology, Inc., Beverly, MA); 2) anti-Akt rabbit polyclonal antibody, which recognizes total Akt (1:2000, Cell Signaling Technology); 3) anti-phospho GSK-3β rabbit polyclonal antibody, which recognizes GSK-3β phosphorylated at Ser9 (1:1000; Cell Signaling Technology); 4) anti-GSK-3β mouse monoclonal antibody, which recognizes total GSK-3β (1:1000, Biosource International, Inc., Camarillo, CA); 5) anti-phospho FOXO3A (FKHRL1) rabbit polyclonal antibody, which recognizes FOXO3A phosphorylated at Ser253 (1:1000; Upstate Biotechnology, Inc.); 6) anti-FOXO3A rabbit polyclonal antibody, which recognizes total FOXO3A (1:1000, Upstate Biotechnology, Inc.); 7) anti-phospho MAPK (p-ERK1/2) mouse monoclonal antibody, which recognizes ERK1 and ERK2 phosphorylated on both at Thr202 and Tyr204 residues (clone 12D4, 1:5000, Upstate Biotechnology, Inc., Lake Placid, NY); 8) anti-MAPK1/2 (ERK1/2) rabbit polyclonal antibody, which recognizes total ERK1/ERK2 (1:5000, Upstate Biotechnology, Inc.); 9) anti-β-actin mouse monoclonal antibody, which recognizes an epitope located within the N-terminal domain of the β-isoform of actin (1:20000; Sigma, Saint Louis, MI). Secondary antibodies for Westerns were horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:5000, Amersham, Buckinghamshire, England, UK) for polyclonal antibodies, or sheep anti-mouse IgG (1:2500, Amersham) for monoclonal antibodies.

4.8. Caspase activity assay

Caspase activity assays were performed on fresh frozen brain sections using the APO LOGIXTM carboxy-fluorescein (FAM) caspase detection kit (Cell Technology, Minneapolis, MN) according to manufacturer’s instructions. FAM-DEVD-FMK is a carboxy-fluorescein-tagged analog of zDEVD-fluoromethyl ketone (FMK), a broad-spectrum cysteine protease inhibitor that enters cells and irreversibly binds activated caspases (Amstad et al., 2001; Bedner et al., 2000; Smolewski et al., 2001). FAM-DEVD-FMK exhibits higher affinity for caspase-3 than for caspase-8, caspase-7, caspase-10 or caspase-6 (Garcia-Calvo et al., 1998) and exhibits much lower affinity for the calpains than for caspases; thus, at 5 μM FAM-DEVD-FMK is a relatively selective inhibitor of caspase-3. Moreover, FAM-DEVD-FMK labeling of CA1 neurons correlates well with caspase-3 activation, as assessed by Western blot analysis. In this study we therefore refer to FAM-DEVD-FMK labeling as indicative of caspase-3 activity. In brief, estradiol- and vehicle-injected animals were deeply anesthetized with pentobarbital (50 mg/kg, i.p.) and killed by decapitation at 24 h after ischemia or sham operation (control). Brains were removed, frozen and cut into sections (18 μm) in the coronal plane of the dorsal hippocampus. Brain sections (3 per animal) were labeled with 5 μM FAM-DEVD-FMK (1 h, 37°C), washed three times with 1× Working Dilution Wash Buffer and viewed under a Nikon ECLIPSE TE-300 fluorescent microscope equipped with an image analysis system at an excitation wavelength of 488 nm and emission wavelength of 565 nm. Images were acquired with a SPOT RT CCD-cooled camera with Diagnostic Software version 3.0. For quantitation of caspase-3 activity, the fluorescence intensity within the entire hippocampal CA1 cell layer was analyzed using NIH Image 1.61. The mean fluorescence intensity of CA1 in the right and left hemisphere from each of the three sections was averaged to provide a single value for each animal.

4.9. Statistical analysis

The results were expressed as mean ± SEM. Data analysis was performed using GraphPad Prism 4.00. Statistical comparisons among groups were conducted using a two-way ANOVA with Bonferroni’s multiple comparisons or t-test post hoc analysis (neuron counts, immunoblots and caspase-3 activity). T-test was used for the serum estradiol data. Differences were considered significant at P < 0.05.

Acknowledgments

Supported by NIH grant NS045693 (to R.S.Z), American Heart Association Development Award 0335285N (to T.J-M.), ISCIII RETICS-RENEVAS grant RD06/0026/0006 (to T.J-M. and E.A.) and the F.M. Kirby Program in Neuroprotection and Repair. The authors thank Nicolas Bamat and Fabrizio Pontarelli for excellent technical assistance.

Abbreviations

Akt
protein kinase B
CREB
cAMP response element binding protein
ERK
extra-cellular-regulated kinases
FOXO
forkhead transcription factor of the O class
GSK3β
glycogen synthase kinase-3β
MAPK
mitogen-activated protein kinase
PI3K
phosphoinositide 3-kinase

Footnotes

Section: 1. Cellular and Molecular Biology of Nervous Systems

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REFERENCES

  • Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 1996;15:6541–6551. [PMC free article] [PubMed]
  • Amantea D, Russo R, Bagetta G, Corasaniti MT. From clinical evidence to molecular mechanisms underlying neuroprotection afforded by estrogens. Pharmacol. Res. 2005;52:119–132. [PubMed]
  • Amstad PA, Yu G, Johnson GL, Lee BW, Dhawan S, Phelps DJ. Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. Biotechniques. 2001;31:608–10. 612, 614. passim. [PubMed]
  • Bedner E, Smolewski P, Amstad P, Darzynkiewicz Z. Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Res. 2000;259:308–313. [PubMed]
  • Behl C. Oestrogen as a neuroprotective hormone. Nature Reviews. 2002;3:433–442. [PubMed]
  • Bi R, Broutman G, Foy MR, Thompson RF, Baudry M. The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus. Proc. Natl. Acad. Sci. USA. 2000;97:3602–3607. [PMC free article] [PubMed]
  • Blevins JE, Stanley BG, Reidelberger RD. DMSO as a vehicle for central injections: tests with feeding elicited by norepinephrine injected into the paraventricular nucleus. Pharmacol. Biochem. Behav. 2002;71:277–282. [PubMed]
  • Bredesen DE. Programmed cell death mechanisms in neurological disease. Curr. Mol. Med. 2008;8:173–186. [PubMed]
  • Brunet A, Datta SR, Greenberg ME. Transcription-dependent and - independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr. Opin. Neurobiol. 2001;11:297–305. [PubMed]
  • Bryant DN, Sheldahl LC, Marriott LK, Shapiro RA, Dorsa DM. Multiple pathways transmit neuroprotective effects of gonadal steroids. Endocrine. 2006;29:199–207. [PubMed]
  • Calderone A, Jover T, Noh K-M, Tanaka H, Yokota H, Lin Y, Grooms S, Regis R, Bennett MV, Zukin RS. Ischemic insults de-repress the gene silencer rest in neurons destined to die. J. Neurosci. 2003;23:2112–2121. [PubMed]
  • Cardona-Gomez GP, Mendez P, DonCarlos LL, Azcoitia I, Garcia-Segura LM. Interactions of estrogen and insulin-like growth factor-I in the brain: molecular mechanisms and functional implications. J. Steroid Biochem. Mol. Biol. 2002a;83:211–217. [PubMed]
  • Cardona-Gomez GP, Mendez P, Garcia-Segura LM. Synergistic interaction of estradiol and insulin-like growth factor-I in the activation of PI3K/Akt signaling in the adult rat hypothalamus. Brain Res. Mol. Brain Res. 2002b;107:80–88. [PubMed]
  • Cimarosti H, Siqueira IR, Zamin LL, Nassif M, Balk R, Frozza R, Dalmaz C, Netto CA, Salbego C. Neuroprotection and protein damage prevention by estradiol replacement in rat hippocampal slices exposed to oxygen-glucose deprivation. Neurochem. Res. 2005a;30:583–589. [PubMed]
  • Cimarosti H, Zamin LL, Frozza R, Nassif M, Horn AP, Tavares A, Netto CA, Salbego C. Estradiol protects against oxygen and glucose deprivation in rat hippocampal organotypic cultures and activates Akt and inactivates GSK-3beta. Neurochem. Res. 2005b;30:191–199. [PubMed]
  • Cordoba Montoya DA, Carrer HF. Estrogen facilitates induction of long term potentiation in the hippocampus of awake rats. Brain Res. 1997;778:430–438. [PubMed]
  • Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995;378:785–789. [PubMed]
  • Crowder RJ, Freeman RS. Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal. J. Biol. Chem. 2000;275:34266–34271. [PubMed]
  • Cyr M, Ghribi O, Thibault C, Morissette M, Landry M, Di PT. Ovarian steroids and selective estrogen receptor modulators activity on rat brain NMDA and AMPA receptors. Brain Res. Brain Res. Rev. 2001;37:153–161. [PubMed]
  • Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999;13:2905–2927. [PubMed]
  • Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997;91:231–241. [PubMed]
  • Endo H, Nito C, Kamada H, Nishi T, Chan PH. Activation of the Akt/GSK3beta signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rats. J. Cereb. Blood Flow Metab. 2006;26:1479–1489. [PubMed]
  • Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW, Thornberry NA. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 1998;273:32608–32613. [PubMed]
  • Garcia-Segura LM, Azcoitia I, DonCarlos LL. Neuroprotection by estradiol. Prog. Neurobiol. 2001;63:29–60. [PubMed]
  • Gazzaley AH, Weiland NG, McEwen BS, Morrison JH. Differential regulation of NMDAR1 mRNA and protein by estradiol in the rat hippocampus. J. Neurosci. 1996;16:6830–6838. [PubMed]
  • Gould E, Woolley CS, Frankfurt M, McEwen BS. Gonadal steroids regulate dendritic spine density in hippocampal pyramidal cells in adulthood. J. Neurosci. 1990;10:1286–1291. [PubMed]
  • Graham SH, Chen J. Programmed cell death in cerebral ischemia. J. Cereb. Blood Flow Metab. 2001;21:99–109. [PubMed]
  • Gu Q, Moss RL. Novel mechanism for non-genomic action of 17 beta-oestradiol on kainate-induced currents in isolated rat CA1 hippocampal neurones. J. Physiol. 1998;506(Pt 3):745–754. [PMC free article] [PubMed]
  • Gulinello M, Lebesgue D, Jover-Mengual T, Zukin RS, Etgen AM. Acute and chronic estradiol treatments reduce memory deficits induced by transient global ischemia in female rats. Horm. Behav. 2006;49:246–260. [PMC free article] [PubMed]
  • Gupta RR, Sen S, Diepenhorst LL, Rudick CN, Maren S. Estrogen modulates sexually dimorphic contextual fear conditioning and hippocampal long-term potentiation (LTP) in rats(1) Brain Res. 2001;888:356–365. [PubMed]
  • Hao J, Janssen WG, Tang Y, Roberts JA, McKay H, Lasley B, Allen PB, Greengard P, Rapp PR, Kordower JH, Hof PR, Morrison JH. Estrogen increases the number of spinophilin-immunoreactive spines in the hippocampus of young and aged female rhesus monkeys. J. Comp Neurol. 2003;465:540–550. [PubMed]
  • Harms C, Lautenschlager M, Bergk A, Katchanov J, Freyer D, Kapinya K, Herwig U, Megow D, Dirnagl U, Weber JR, Hortnagl H. Differential mechanisms of neuroprotection by 17 beta-estradiol in apoptotic versus necrotic neurodegeneration. J. Neurosci. 2001;21:2600–2609. [PubMed]
  • Honda K, Sawada H, Kihara T, Urushitani M, Nakamizo T, Akaike A, Shimohama S. Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons. J. Neurosci. Res. 2000;60:321–327. [PubMed]
  • Honda K, Shimohama S, Sawada H, Kihara T, Nakamizo T, Shibasaki H, Akaike A. Nongenomic antiapoptotic signal transduction by estrogen in cultured cortical neurons. J. Neurosci. Res. 2001;64:466–475. [PubMed]
  • Jover T, Tanaka H, Calderone A, Oguro K, Bennett MV, Etgen AM, Zukin RS. Estrogen protects against global ischemia-induced neuronal death and prevents activation of apoptotic signaling cascades in the hippocampal CA1. J. Neurosci. 2002;22:2115–2124. [PubMed]
  • Jover-Mengual T, Zukin RS, Etgen AM. MAPK signaling is critical to estradiol protection of CA1 neurons in global ischemia. Endocrinology. 2007;148:1131–1143. [PMC free article] [PubMed]
  • Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R, Grohe C. Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway. J. Biol. Chem. 2000;275:18447–18453. [PubMed]
  • Kawano T, Morioka M, Yano S, Hamada J, Ushio Y, Miyamoto E, Fukunaga K. Decreased akt activity is associated with activation of forkhead transcription factor after transient forebrain ischemia in gerbil hippocampus. J. Cereb. Blood Flow Metab. 2002;22:926–934. [PubMed]
  • Koh PO, Cho GJ, Choi WS. 17beta-estradiol pretreatment prevents the global ischemic injury-induced decrease of Akt activation and bad phosphorylation in gerbils. J. Vet. Med. Sci. 2006;68:1019–1022. [PubMed]
  • Koh SH, Kim SH, Kwon H, Park Y, Kim KS, Song CW, Kim J, Kim MH, Yu HJ, Henkel JS, Jung HK. Epigallocatechin gallate protects nerve growth factor differentiated PC12 cells from oxidative-radical-stress-induced apoptosis through its effect on phosphoinositide 3-kinase/Akt and glycogen synthase kinase-3. Brain Res. Mol. Brain Res. 2003;118:72–81. [PubMed]
  • Lebesgue D, Chevaleyre V, Zukin RS, Etgen AM. Estradiol rescues neurons from global ischemia-induced cell death: multiple cellular pathways of neuroprotection. Steroids. 2009;74:555–561. [PMC free article] [PubMed]
  • Liou AK, Clark RS, Henshall DC, Yin XM, Chen J. To die or not to die for neurons in ischemia, traumatic brain injury and epilepsy: a review on the stress-activated signaling pathways and apoptotic pathways. Prog. Neurobiol. 2003;69:103–142. [PubMed]
  • Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunities in stroke. Nat. Rev. Neurosci. 2003;4:399–415. [PubMed]
  • McEwen B. Estrogen actions throughout the brain. Recent Prog. Horm. Res. 2002;57:357–384. [PubMed]
  • Mendez P, Azcoitia I, Garcia-Segura LM. Estrogen receptor alpha forms estrogen-dependent multimolecular complexes with insulin-like growth factor receptor and phosphatidylinositol 3-kinase in the adult rat brain. Brain Res Mol. Brain Res. 2003;112:170–176. [PubMed]
  • Mendez P, Garcia-Segura LM. Phosphatidylinositol 3-kinase and glycogen synthase kinase 3 regulate estrogen receptor-mediated transcription in neuronal cells. Endocrinology. 2006;147:3027–3039. [PubMed]
  • Miller NR, Jover T, Cohen HW, Zukin RS, Etgen AM. Estrogen can act via estrogen receptor alpha and beta to protect hippocampal neurons against global ischemia-induced cell death. Endocrinology. 2005;146:3070–3079. [PubMed]
  • Miyawaki T, Mashiko T, Ofengeim D, Flannery RJ, Noh KM, Fujisawa S, Bonanni L, Bennett MV, Zukin RS, Jonas EA. Ischemic preconditioning blocks BAD translocation, Bcl-xL cleavage, and large channel activity in mitochondria of postischemic hippocampal neurons. Proc. Natl. Acad. Sci. U. S. A. 2008;105:4892–4897. [PMC free article] [PubMed]
  • Miyawaki T, Ofengeim D, Noh KM, Latuszek-Barrantes A, Hemmings BA, Follenzi A, Zukin RS. The endogenous inhibitor of Akt, CTMP, is critical to ischemia-induced neuronal death. Nat. Neurosci. 2009;12:618–626. [PMC free article] [PubMed]
  • Moss RL, Gu Q. Estrogen: mechanisms for a rapid action in CA1 hippocampal neurons. Steroids. 1999;64:14–21. [PubMed]
  • Namura S, Nagata I, Kikuchi H, Andreucci M, Alessandrini A. Serine-threonine protein kinase Akt does not mediate ischemic tolerance after global ischemia in the gerbil. J. Cereb. Blood Flow Metab. 2000;20:1301–1305. [PubMed]
  • Nishimoto T, Kihara T, Akaike A, Niidome T, Sugimoto H. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate attenuates glutamate-induced caspase-3 cleavage via regulation of glycogen synthase kinase 3beta. J. Neurosci. Res. 2008;86:1096–1105. [PubMed]
  • Noshita N, Lewen A, Sugawara T, Chan PH. Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice. J. Cereb. Blood Flow Metab. 2001;21:1442–1450. [PubMed]
  • Noshita N, Sugawara T, Lewen A, Hayashi T, Chan PH. Copper-zinc superoxide dismutase affects Akt activation after transient focal cerebral ischemia in mice. Stroke. 2003;34:1513–1518. [PubMed]
  • Paxinos GAWC. The rat brain in stereotaxic coordinates. Academic Press; New York: 1998.
  • Plamondon H, Morin A, Charron C. Chronic 17beta-estradiol pretreatment and ischemia-induced hippocampal degeneration and memory impairments: a 6-month survival study. Horm. Behav. 2006;50:361–369. [PubMed]
  • Pozzo-Miller LD, Inoue T, Murphy DD. Estradiol increases spine density and NMDA-dependent Ca2+ transients in spines of CA1 pyramidal neurons from hippocampal slices. J. Neurophysiol. 1999;81:1404–1411. [PubMed]
  • Prange-Kiel J, Rune GM. Direct and indirect effects of estrogen on rat hippocampus. Neuroscience. 2006;138:765–772. [PubMed]
  • Raval AP, Saul I, Dave KR, DeFazio RA, Perez-Pinzon MA, Bramlett H. Pretreatment with a single estradiol-17beta bolus activates cyclic-AMP response element binding protein and protects CA1 neurons against global cerebral ischemia. Neuroscience. 2009;160:307–318. [PMC free article] [PubMed]
  • Saito A, Hayashi T, Okuno S, Ferrand-Drake M, Chan PH. Overexpression of copper/zinc superoxide dismutase in transgenic mice protects against neuronal cell death after transient focal ischemia by blocking activation of the Bad cell death signaling pathway. J. Neurosci. 2003;23:1710–1718. [PubMed]
  • Sandstrom NJ, Rowan MH. Acute pretreatment with estradiol protects against CA1 cell loss and spatial learning impairments resulting from transient global ischemia. Horm. Behav. 2007;51:335–345. [PMC free article] [PubMed]
  • Sato K, Akaishi T, Matsuki N, Ohno Y, Nakazawa K. beta-Estradiol induces synaptogenesis in the hippocampus by enhancing brain-derived neurotrophic factor release from dentate gyrus granule cells. Brain Res. 2007;1150:108–120. [PubMed]
  • Segars JH, Driggers PH. Estrogen action and cytoplasmic signaling cascades. Part I: membrane-associated signaling complexes. Trends Endocrinol. Metab. 2002;13:349–354. [PMC free article] [PubMed]
  • Smith CC, McMahon LL. Estrogen-induced increase in the magnitude of long-term potentiation occurs only when the ratio of NMDA transmission to AMPA transmission is increased. J. Neurosci. 2005;25:7780–7791. [PubMed]
  • Smith CC, McMahon LL. Estradiol-induced increase in the magnitude of long-term potentiation is prevented by blocking NR2B-containing receptors. J. Neurosci. 2006;26:8517–8522. [PubMed]
  • Smolewski P, Bedner E, Du L, Hsieh TC, Wu JM, Phelps DJ, Darzynkiewicz Z. Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry. Cytometry. 2001;44:73–82. [PubMed]
  • Soderstrom I, Strand M, Ingridsson AC, Nasic S, Olsson T. 17beta-estradiol and enriched environment accelerate cognitive recovery after focal brain ischemia. Eur. J. Neurosci. 2009;29:1215–1224. [PubMed]
  • Srivastava DP, Woolfrey KM, Jones KA, Shum CY, Lash LL, Swanson GT, Penzes P. Rapid enhancement of two-step wiring plasticity by estrogen and NMDA receptor activity. Proc. Natl. Acad. Sci. U. S. A. 2008;105:14650–14655. [PMC free article] [PubMed]
  • Sweatt JD. Mitogen-activated protein kinases in synaptic plasticity and memory. Curr. Opin. Neurobiol. 2004;14:311–317. [PubMed]
  • Tanaka H, Yokota H, Jover T, Cappuccio I, Calderone A, Simionescu M, Bennett MV, Zukin RS. Ischemic preconditioning: neuronal survival in the face of caspase-3 activation. J. Neurosci. 2004;24:2750–2759. [PubMed]
  • Thomas GM, Huganir RL. MAPK cascade signalling and synaptic plasticity. Nat. Rev. Neurosci. 2004;5:173–183. [PubMed]
  • Wise P. Estradiol exerts neuroprotective actions against ischemic brain injury: insights derived from animal models. Endocrine. 2003;21:11–15. [PubMed]
  • Won CK, Ji HH, Koh PO. Estradiol prevents the focal cerebral ischemic injury-induced decrease of forkhead transcription factors phosphorylation. Neurosci. Lett. 2006;398:39–43. [PubMed]
  • Woolley CS, McEwen BS. Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat. J. Neurosci. 1992;12:2549–2554. [PubMed]
  • Woolley CS, McEwen BS. Estradiol regulates hippocampal dendritic spine density via an N-methyl-D-aspartate receptor-dependent mechanism. J. Neurosci. 1994;14:7680–7687. [PubMed]
  • Yano S, Morioka M, Fukunaga K, Kawano T, Hara T, Kai Y, Hamada J, Miyamoto E, Ushio Y. Activation of Akt/protein kinase B contributes to induction of ischemic tolerance in the CA1 subfield of gerbil hippocampus. J. Cereb. Blood Flow Metab. 2001;21:351–360. [PubMed]
  • Zukin RS, Jover T, Yokota H, Calderone A, Simionescu M, Lau CG. Molecular and Cellular Mechanisms of Ischemia-induced Neuronal Death. In: Mohr JP, Choi D, Grotta JC, Weir B, Wolf PA, editors. Stroke: Pathophysiology, Diagnosis, and Management. Churchill Livingstone; Philadelphia: 2004. pp. 829–854.
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