17β-Estradiol attenuates secondary injury through activation of Akt signaling via estrogen receptor alpha in rat brain following subarachnoid hemorrhage

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) e 2 3 ee 3 0

Available online at www.sciencedirect.com

journal homepage: www.JournalofSurgicalResearch.com

17b-Estradiol attenuates secondary injury through activation of Akt signaling via estrogen receptor alpha in rat brain following subarachnoid hemorrhage Cheng-Hsing Kao, MD,a,b Chih-Zen Chang, MD,c,d,e Yu-Feng Su, MD,c,d Yee-Jean Tsai, MS,c Kao-Ping Chang, MD,d,f Tzu-Kang Lin, MD,g Shiuh-Lin Hwang, MD, PhD,c,d and Chih-Lung Lin, MD, PhDc,d,* a

Center for General Education, Southern Taiwan University of Technology, Tainan, Taiwan Department of Neurosurgery, Chi Mei Medical Center, Tainan, Taiwan c Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan d Faculty of Medicine, Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan e Department of Surgery, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan, Republic of China f Division of Plastic and Reconstructive Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan g Department of Neurosurgery, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan b

article info

abstract

Article history:

Background: Apoptosis is implicated in vasospasm and the long-term sequelae of

Received 25 July 2012

subarachnoid hemorrhage (SAH). This study tested the hypothesis that attenuation of

Received in revised form

SAH-induced apoptosis after 17b-estradiol (E2) treatment is associated with an increase in

12 January 2013

phosphorylation of Akt via estrogen receptor-a (ER-a) in rats.

Accepted 17 January 2013

Materials and methods: We examined the expression of phospho-Akt, ERa and ERb, and

Available online 8 February 2013

apoptosis in cerebral cortex, hippocampus, and dentate gyrus in a two-hemorrhage SAH model in rats. We subcutaneously implanted other rats with a silicone rubber tube con-

Keywords:

taining E2; they received daily injections of nonselective estrogen receptor antagonist (ICI

Estrogen

182,780), selective ERa-selective antagonist (methyl-piperidino-pyrazole), or ERb-selective

Estrogen receptor-a

antagonist (R,R-tetrahydrochrysene) after the first hemorrhage.

Subarachnoid hemorrhage

Results: At 7 d after the first SAH, protein levels of phospho-Akt and ERa were significantly

Cerebral vasospasm

decreased and caspase-3 was significantly increased in the dentate gyrus. The cell death

PI3K/Akt

assay revealed that DNA fragmentation was significantly increased in the dentate gyrus. Those actions were reversed by E2 and blocked by ICI 182,780 and methyl-piperidinopyrazole, but not R,R-tetrahydrochrysene. However, there were no significant changes in the expression of the protein levels of phospho-Akt, ERa, ERb, and caspase-3, and DNA fragmentation after SAH. Conclusions: The present study shows that a beneficial effect of E2 in attenuating SAHinduced apoptosis is associated with activation of the expression of phospho-Akt and ERa, and alteration in caspase-3 protein expression via an ERa-dependent mechanism in the dentate gyrus. These data support further the investigation of E2 in the treatment of SAH in humans. ª 2013 Elsevier Inc. All rights reserved.

* Corresponding author. Department of Neurosurgery, Kaohsiung Medical University Hospital, No. 100 Tz-you 1st Road, Koahsiung City 807, Taiwan. Tel.: þ886 7 3121101 extension 5880; fax: þ886 7 3215049. E-mail address: [email protected] (C.-L. Lin). 0022-4804/$ e see front matter ª 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.01.033

e24

1.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) e 2 3 ee 3 0

Introduction

Although cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) has been recognized for more than half a century, its pathophysiologic mechanism remains illusive and no effective treatment for cerebral vasospasm exists to date [1]. Apoptosis of granule cells in the dentate gyrus, which is a frequent observation in several animal models of the disease, is detected in patients dying of SAH [2]. It has also been shown that apoptosis in the vasculature has a significant role in SAH, and that the apoptotic cascades may be responsible for vasospasm [3,4]. Furthermore, cell death after SAH has important implications not only for vasospasm, but also as the long-term sequelae of SAH [3]. Our previous studies have revealed that 17b-estradiol (E2) could attenuate experimental SAH-induced cerebral vasospasm by inhibiting endothelin-1 production [5], preventing the augmentation of inducible nitric oxide synthase expression, and preserving the normal endothelial nitric oxide synthase (eNOS) expression after SAH [6]. The prevention of increased inducible nitric oxide synthase expression is achieved by interfering with nuclear factor-kB (NF-kB) transactivation via an estrogen receptor (ER)-dependent mechanism [7]. Our other findings also demonstrated that E2 reverses the decreased expression of adenosine A1 receptors (AR-A1) and increases the expression of adenosine A2A receptors (AR- A2A) to prevent vasospasm and apoptosis induced by SAH in the dentate gyrus [8e11]. Concerning the role of female sex hormone E2 in the longterm sequelae of SAH, especially related to apoptosis, its influence has yet to be determined [12]. Recent studies have shown that estrogen offers neuroprotection against both neurodegenerative diseases and brain injury [13]. This was previously revealed in an animal study, in which estrogen was effective in attenuating neuronal death after focal and global ischemia [14]. Clinically, estrogen replacement therapy in postmenopausal women reduces the incidence of stroke, the extent of ischemic neurodegeneration [15], and the onset and severity of Alzheimer disease [16]. Akt, a serine/threonine protein kinase also known as proteinase B, has an important role in the cell death/survival pathway in multiple cell lineages by inhibiting apoptosis [17,18]. It is activated by phosphorylation at the Ser473 residue and acts downstream of the phosphoinositide 3-kinase (PI3-K) pathway [17]. To begin with, PI3-K is responsible for the recruitment of Akt to the cellular membrane and the activation of Akt [19]. Upon activation, PI3-K then induces Akt phosphorylation, which in turn phosphorylates and blocks the actions of BAD, a proapoptotic member of the Bcl-2 family [18]. Besides, PI3-K also alters the activities of a number of other proapoptotic mediators, including caspase-9, forkhead, and NF-kB [20e22]. Moreover, activated Akt also phosphorylates and inhibits downstream substrates, including glycogen synthase kinase-3b (GSK3b) [23], which leads to neuronal resistance to apoptotic stimuli through the promotion of cell survival and the suppression of apoptosis [24,25]. Currently, the Akt survival pathway in cerebral ischemia, traumatic brain injury, spinal cord injury, and SAH has been established to be involved in the mechanisms of apoptotic neuronal death

[26e30]. However, the role of Akt survival pathway in the antiapoptotic effect of E2 against brain injury has not been studied. In the present study, we aimed to clarify the following points: (1) whether SAH leads to apoptosis in rat brain in the two-hemorrhage SAH model; whether SAH down-regulates estrogen receptor a (ERa) and phospho-Akt in rat brain after SAH; whether E2 activates ERa and phospho-Akt and alternates the activity of caspase-3; and whether E2 has an antiapoptotic effect against brain injury after SAH via an ERadependent mechanism.

2.

Materials and methods

2.1.

Animal preparation

The Kaohsiung Medical University Animal Research Committee approved all experimental protocols. We used as subjects male Sprague-Dawley rats (Education Research Resource, National Laboratory Animal Center, Taiwan), weighing approximately 338e389 g. We kept these experimental rats on a 12-h light/dark cycle, with free access to food and water. They were evenly divided into seven groups. Rats in group 1 served as controls and were not subjected to SAH (control; n ¼ 12). The rats in all other groups were subjected to experimental SAH, as described below. Rats in group 2 received experimental SAH without additional treatment (SAH only; n ¼ 12). Rats in group 3 received experimental SAH with vehicle treatment (SAH plus vehicle; n ¼ 12). Rats in group 4 received experimental SAH with E2 treatment (SAH plus E2; n ¼ 12). We delivered E2 by subcutaneous implantation of a 30-mm-long silicone rubber tube with a 2-mm inner diameter and 4-mm outer diameter (Shin-Etsu Polymer Co, Ltd, Japan) containing 0.3 mg/mL 17b-estradiol benzoate in corn oil (Sigma), giving rise to the physiologic range of plasma E2 concentrations (56e92 pg/mL) [6]. Rats in group 5 received experimental SAH with E2 and ICI 182,780 (a nonselective ER antagonist) (Tocris, UK) treatment (SAH plus E2 plus ICI; n ¼ 12). We gave ICI 182,780 (2 mg/kg/d, intraperitoneally) for 7 d after the first hemorrhage. We dissolved ICI 182,780 in corn oil (Sigma) to make a final 20% solution. To study the involvement of ERa or ERb in E2-mediated antiapoptosis, we treated rats in group 6 (SAH plus E2 plus methyl-piperidino-pyrazole [MPP]; n ¼ 12) and group 7 (SAH plus E2 plus R,R-tetrahydrochrysene [R,RTHC]; n ¼ 12) with E2. They received daily subcutaneous injection of ERa-selective antagonist (MPP, 2.0 mg/kg) (Tocris) or ERb-selective antagonist (R,R-THC, 0.1 mg/kg) (Tocris) for 7 d after the first hemorrhage. We modified the antagonizing dose of MPP from a mouse study by Davis et al. [31] and modified the relative antagonizing dose for R,R-THC from a microinjection study in rats proposed by Gingerich and Krukoff [32].

2.2.

Induction of experimental SAH

We anesthetized the rats by an intraperitoneal injection of pentobarbital (50 mg/kg). We fixed the head of each animal in

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) e 2 3 ee 3 0

a stereotactic frame. Then, we punctured the cisterna magna percutaneously using a 25-gauge butterfly needle. We slowly withdrew about 0.1e0.15 mL cerebrospinal fluid. After we drew the first sample, we clamped the junction of the butterfly needle and tube. Afterward, we withdrew 0.3 mL freshly autologous nonheparinized blood from the tail artery. Using a needle-in-needle method (inserting a 30-gauge needle into a 25-gauge butterfly needle at the junction of needle and tube), the nonheparinized blood previously drawn from the tail was injected slowly into the cisterna magna in approximately 2 min [6]. We repeated the procedure 48 h later. Hence, in this study, the term “SAH” refers to an experimental model of SAH produced by double injection of nonheparinized blood into the cisterna magna (two-hemorrhage SAH model). At 7 d after the first SAH, we killed the animals by perfusion. Subsequently, we removed the brain, dissected it, and then stored it at 80 C.

2.3.

Cell death assay

We included six animals in each group in this protocol. We used a commercial enzyme immunoassay to determine cytoplasmic histone-associated DNA fragments (Roche, Mannheim, Germany) to quantify the DNA fragmentation, which indicates apoptotic cell death. We took fresh brain tissuedcortex, hippocampus (CA1, CA2, and CA3), and dentate gyrusdand extracted protein via cytosolic fraction in nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL). According to the manufacturer’s protocol, we used a cytosolic volume containing 300 mg protein for the enzyme-linked immunosorbent assay.

2.4.

Protein extraction

We included six animals from each group in this part of the study. Samples were obtained by dissection from the cerebral cortex, hippocampus (CA1, CA2, and CA3), and dentate gyrus under the operating microscope. We homogenized brain tissue in ice-cold lysis buffer: mammalian protein extraction reagent (Pierce) with protease inhibitor (Complete Mini; Roche). We centrifuged tissue lysates at 15,000 rpm for 20 min at 4 C, then collected the suspension. The protein concentration was analyzed by a protein assay kit based on the Bradford method (Bio-Rad Laboratories, Hercules, CA). Proteins were stored at 80 C.

2.5.

Measurement caspase-3 activity

We measured caspase-3 activity using a caspase-3 colorimetric assay kit (Assay Designs, Ann Arbor, MI), to monitor apoptosis in brain homogenates. We used tissue lysates (100 g) for caspase-3 activity. As the AcDEVD-p-nitroaniline substrate was cleaved by activated caspase-3, we achieved the detection of liberated p-nitroaniline at 405 nm in a spectrophotometer equipped for 96-well microtiter plates. Caspase-3 activities are reported as units of caspase-3 per milligram protein. In this assay design, one unit of Caspase-3 is defined as the release of 1 pmol p-nitroaniline/min from the 50-mmol/L AcDEVD-p-nitroaniline substrate at 3 C.

2.6.

e25

Western blot analysis

We resolved tissue lysates (30 g) on 10% sodium dodecyl sulfateepolyacrylamide gel electrophoresis (Bio-Rad Laboratories). We transferred the gels onto polyvinylidene difluoride (PerkinElmer, Waltham, MA) membrane using electroblotting for 90 min (100 V). The membrane was blocked overnight at 4 C with Tween-Tris buffer saline solution (t-TBS; 20 mmol/L Tris base, 0.44 mmol/L NaCl, 0.1% Tween 20, pH 7.6), which contains 5% nonfat dry milk. We probed the membrane with primary antibodies ERa (1:500; Affinity BioReagents, Golden, CO), ERb (1:1000; Affinity BioReagents), phospho-Akt (1:1000; Cell Signaling Technology, Inc., Beverly, MA), Akt (1:4000; Cell Signaling Technology, Inc.), and b-actin (1:80,000; SigmaAldrich, St. Louis, MO) overnight at 4 C. Then, we rinsed it with t-TBS for 10 min three times, and incubated it with a 1:2000 dilution of horseradish peroxidaseeconjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). In this study, Akt and b-actin levels served as internal standards to account for the loading differences. We rinsed the membranes with t-TBS for 10 min three times, and incubated them with Enhanced Chemiluminescence Reagent (PerkinElmers) to visualize the signal before exposure to the Fuji medical x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan). For more accuracy, we performed the experiment in triplicate. We scanned the x-ray films and the determined optical density by employing ImageJ (National Institutes of Health, Bethesda, MD).

2.7.

Data analysis

Data are expressed as mean  standard deviation (SD). We analyzed them by one-way analysis of variance followed by Scheffe´’s post hoc test. Significance was accepted at P < 0.05.

3.

Results

Before perfusion-fixation, there were no significant differences among the seven groups in the physiologic parameters, including body weight, arterial blood pH, PaCO2, PaO2, and mean arterial blood pressure. We observed a thick subarachnoid clot over the basal surface of the brain stem in each animal subjected to SAH. The mortality rate at 7 d after the first SAH was 0%.

3.1. Activation of ERa involved in the antiapoptotic effect of E2 in the dentate gyrus after SAH We analyzed DNA fragmentation with a commercial enzyme immunoassay (cell death assay) 7 d after the first SAH (Fig. 1). The cell death assay revealed no significant difference in DNA fragmentation in cortex and hippocampus between control and SAH animals with or without treatment, but was significantly increased in the dentate gyrus after SAH in the SAHonly group and SAH plus vehicle group (Fig. 1). Compared with the SAH-only and SAH plus vehicle groups, DNA fragmentation in the SAH plus E2 group decreased significantly (P < 0.01). On the one hand, pretreatment of the rats with ICI 182,780 and ERa-selective antagonist MPP reversed the

e26

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) e 2 3 ee 3 0

Fig. 1 e 17b-Estradiol prevented cell death in the dentate gyrus after SAH. The cell death assay revealed no significant difference in DNA fragmentation in cortex and hippocampus between control and SAH animals, but significantly increased in the dentate gyrus after SAH in the SAH-only group and SAH plus vehicle group. Compared with the SAH-only and SAH plus vehicle groups, DNA fragmentation in the SAH plus E2 group decreased significantly (P < 0.01). Pretreatment of rats with ICI 182,780 and MPP but not R,R-THC reversed the E2-mediated prevention of apoptosis. n [ 5 in each group; ***P < 0.001, significantly different from the control group; **P < 0.01, significantly different from the control group; ##P < 0.01, significantly different from the SAH-only and SAH plus vehicle groups.

E2-mediated prevention of apoptosis. On the other hand, pretreatment of the rats with R,R-THC did not reverse E2mediated prevention of SAH-induced apoptosis (Fig. 1). These results indicate that (1) apoptotic cell death increased in the dentate gyrus 7 d after SAH; (2) E2 treatment has an antiapoptotic effect in the dentate gyrus, but not completely; and (3) activation of ERa, but not ERb, was involved in the E2mediated prevention of SAH-induced apoptosis.

3.2. 17b-Estradiol prevented SAH-induced brain apoptosis via decreased caspase-3 activity Among the caspases, caspase-3 has emerged as an essential component in several apoptotic pathways. We investigated the activity of casapase-3 using the specific colorimetrically labeled peptide DEVD. The SAH-induced caspase-3 activity increase in the dentate gyrus was significantly higher than in the control (P < 0.05) (Fig. 2). 17b-Estradiol significantly decreased SAH-induced caspase-3 activity in the dentate gyrus, compared with the SAH-only and SAH plus vehicle

groups (P < 0.05). However, we observed no significant effect on caspase-3 activity in the cortex and the hippocampus after SAH in all SAH groups, compared with the control group (P > 0.05). The antiapoptotic effect of E2 in the dentate gyrus was reversed by pretreating animals with ICI 182,780 and MPP, which suggests that activation of ERa was involved in these E2emediated antiapoptotic effects. Pretreatment of the rats with R,R-THC did not reverse E2-mediated prevention of SAHinduced increased caspase-3 activity (Fig. 2). These results suggest that E2 exerts antiapoptotic effects through ERa, whereas ERb may not be involved in the E2-mediated prevention of SAH-induced apoptosis. 17b-Estradiol reversed SAH-induced, down-regulated phospho-Akt protein and ERa expression in the dentate gyrus. There was no significant change in the protein expression of ERb in animals subjected to SAH with or without treatment. To quantify the change in ERa, ERb, and phosphor-Akt protein expression after SAH with or without E2 treatment, we examined protein levels of ERa, ERb, and phospho-Akt in the cortex, hippocampus, and dentate gyrus (Figs. 3e5).

Fig. 2 e 17b-Estradiol prevented caspase-3 activity in the dentate gyrus after SAH. 17b-Estradiol significantly decreased SAH-induced caspase-3 activity in the dentate gyrus, compared with the SAH-only and SAH plus vehicle groups (P < 0.05). However, we observed no significant effect on caspase-3 activity in the cortex and the hippocampus after SAH in all SAH groups, compared with the control group. The antiapoptotic effect of E2 in the dentate gyrus was reversed by ICI 182,780 and MPP but not R,R-THC. n [ 5 in each group; *P < 0.05 compared with control; #P < 0.05 compared with the SAH-only and SAH plus vehicle groups.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) e 2 3 ee 3 0

e27

Fig. 3 e 17b-Estradiol reversed SAH-induced down-regulated ERa expression in the dentate gyrus. 17b-Estradiol treatment reduced the increase in ERa protein expression within the dentate gyrus after SAH. However, we observed no significant effect on ERa protein expression in the cortex and the hippocampus after SAH in all SAH groups, compared with the control group. The effect of E2 on ERa protein expression in the dentate gyrus was reversed by ICI 182,780 and MPP but not R,R-THC. n [ 6 in each group; #P < 0.05, significantly different from the SAH group; *P < 0.05, significantly different from the control group; **P < 0.01, significantly different from the control group.

Quantification showed a decreased expression of ERa and phospho-Akt in the dentate gyrus after SAH, compared with the control group (P < 0.05) (Figs. 3 and 4). 17b-Estradiol treatment increased the expression of ERa and phospho-Akt in the dentate gyrus after SAH, compared with SAH-only and SAH plus vehicle groups (P < 0.05) (Figs. 3 and 4). Pretreatment of the rats with ICI 182,780 or MPP reversed the E2-mediated up-regulation of ERa and phospho-Akt. These results indicate that E2 could reverse SAH down-regulated ERa and phospho-Akt expression via an ERa-dependent mechanism in the dentate gyrus 7 d after SAH. However, there was no significant change in the expression of ERa and phosphoAkt in the cortex and hippocampus between the control and SAH groups with or without E2 treatment. Western blot analysis showed that there was no prominent change in protein levels of ERb in the cortex, hippocampus, and dentate gyrus 7 d after the first SAH, compared with the control group (Fig. 5). 17b-Estradioletreated animals with ICI 182,780, MPP, or R,R-THC had no significant change in the protein expression of ERb.

4.

Discussion

Cognitive dysfunction is a common and disabling sequela of SAH [33]. Apoptotic cell death has been shown to be associated with acute brain injury after SAH [34e39]. Moreover, apoptosis in the dentate gyrus has been observed in human patients with SAH [2]. These reports suggest that apoptotic cell death presents a number of potential therapeutic targets to ameliorate secondary brain injury after SAH. Subarachnoid hemorrhageeinduced apoptosis is cellspecific and has different results, depending on the models and species used [36,40]. Nonetheless, the patterns of distribution of apoptotic cell death after SAH revealed by different studies were not coherent although they used the same SAH model [26,38,39]. In the study by Prunell et al. [39], a noncraniotomy endovascular perforation model resulted in large

variations in the severity of bleeding and a high mortality rate. Our study adopted a two-hemorrhage rat SAH model, and resulted in no mortality of subjects. The occurrence of apoptosis in dental gyrus after SAH is probably attributable to use of the cisternal injection SAH model, which results in prolonged blood clot accumulation. This may explain why we found apoptosis in the dentate gyrus but not the hippocampus or cortex after SAH in our study. Park et al. [38] found that apoptotic changes in most brain regions, especially in the hippocampus, probably result from use of the endovascular filament perforation model, which results in a global ischemia affecting CA1 and the dentate gyrus regions of hippocampus. This discrepancy between previous studies and ours might be the result of the difference in time points for assessing brain damage; and the difference in mechanisms of apoptosis, such as increased intracranial pressure, acute global ischemia, delayed ischemia, acute cerebral flow decrease, and the breakdown of hemoglobulin after SAH. A number of apoptotic pathways have major roles in SAHinduced apoptosis [3]. The Akt signaling pathway has been implicated in neuronal survival in several models of neurodegenerative disease [26,29,30,41]. However, only a few articles studied the role of Akt pathway-mediated cell survival after SAH. Endo et al. [27] reported that the severity of SAHinduced brain injury was correlated with the timing and distribution of phosphorylation of Akt (serine-473) and GSK3b (serine-9). However, the mechanisms of this temporary dephosphorylation of Akt and its influence on subsequent brain injury damage remain to be clarified. Activation of the Akt/GSK3b survival signal pathway improved SAH-induced apoptosis. The neuroprotective effect of simvastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, and copper/zinc-superoxide dismutase resulted from activation of Akt/GSK3b survival signal after SAH [42,43]. Furthermore, Sugawara et al. [44] found that attenuation of cerebral vasospasm after SAH by simvastatin and recombinant erythropoietin was associated with increasing phosphorylation of Akt and eNOS [45]. Our results, which

e28

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) e 2 3 ee 3 0

Fig. 4 e 17b-Estradiol reversed SAH-induced down-regulated phosphor-Akt protein expression in the dentate gyrus. 17bEstradiol treatment reduces the increase in phosphor-Akt protein expression within the dentate gyrus after SAH. However, we observed no significant effect on phosphor-Akt protein expression in the cortex and the hippocampus after SAH in all SAH groups, compared with the control group. The effect of E2 on phosphor-Akt protein expression in the dentate gyrus was reversed by ICI 182,780 and MPP but not R,R-THC. n [ 6 in each group; #P < 0.05, significantly different from the SAH group; ##P < 0.001, significantly different from the SAH group; *P < 0.05, significantly different from the control group; **P < 0.01, significantly different from the control group. showed decreasing phosphorylation of Akt after SAH, which was reversible by E2, further confirm the role of Akt in SAHinduced vasospasm and apoptosis. In this study, we demonstrated that ERa is involved in E2mediated prevention of SAH-induced apoptosis. Estrogenmediated neuroprotection is consistently demonstrated in several oxidative stress models in both cell and animal models [46,47]. In addition, estrogens have broad effects on neuronal plasticity and can modulate apoptosis of dentate granule cells [48,49]. Both receptor-independent and receptor-dependent mechanisms have been proposed to explain its neuroprotection in the brain [50e53]. Estrogen can suppress apoptosis by (1) suppressing the expression of proapoptotic proteins and increasing antiapoptotic proteins [46,54], (2) inhibiting tumor necrosis factor gene transcription, and (3) attenuating TNF-mediated production of chemokine cytokineinduced neutrophil chemoattractant-2b [55e57]. In our study, the protective effect of E2 in the attenuation of SAH-induced

apoptosis was associated with alteration in caspase-3 protein expression via ERa-dependent mechanisms in the dentate gyrus. Considering the expression of ER, it has been previously reported that ERa and ERb might have different roles in the regulation of vascular functions [58]. Estrogen receptor-a, but not ERb, regulates the eNOS pathway to contribute to the effects of estrogen on cerebral artery reactivity [59]. In the present study, we found that the beneficial effect of E2 on attenuating SAH-induced apoptosis is associated with activation of the expression of phospho-Akt and ERa, and alteration in caspaes-3 protein expression via ERa-dependent mechanisms in the dentate gyrus. We previously showed that the beneficial effect of E2 in attenuating SAH-induced vasospasm and apoptosis may result from an increased expression of AR-A2A and ERK via ER-dependent mechanisms [10]. Estrogen can rapidly activate the ERK and PI3K-Akt pathways in cortical and hippocampal cells, effects implicated in

Fig. 5 e Western blot analysis showed no prominent change in protein levels of ERb in the cortex, hippocampus, and dentate gyrus 7 d after the first SAH, compared with the control group. 17b-Estradiol-treated animals with ICI 182,780, MPP, or R,R-THC had no significant change in the protein expression of ERb. n [ 6 in each group.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) e 2 3 ee 3 0

estrogen neuroprotection action [60,61]. The mechanism of the interaction of estrogen with ERK and Akt is unclear, but ER-a can interact with cytoskeleton protein p130Cas in a complex containing Src and PI3K, which are upstream regulators of ERK and Akt activation, respectively [62]. Further studies using the inhibitors of Akt pathway, with AR-A2a and/ or ERK1/2 in the SAH rat model, may offer a better perspective of the relationship among AR-A2a, ERK1/2, and Akt in SAHinduced brain injury, to understand whether these pathways are parallel or sequential in E2-induced protection after SAH. There were no significant changes in expression of the proteins phospho-Akt, ERa, ERb, and caspase-3, and DNA fragmentation after SAH compared with the control group and E2-treated group in the cerebral cortex and hippocampus. 17bEstradiol prevented a focal ischemia-induced decrease in Akt activation and phosphorylation of mTOR and p70S6 kinases, and a subsequent decrease in S6 phosphorylation. On the contralateral cortex without focal ischemia-induced injury, the level of Akt did not change after E2 treatment [63]. Taking these findings together, E2 may activate Akt in neurons subjected to injury. It is possible that E2 does not activate Akt in non-injured neurons in our two-hemorrhage SAH animal model. We found SAH-induced down-regulated phospho-Akt and ERa protein expression in the dentate gyrus in our study. The decrease in phospho-Akt and ERa protein expression can be considered one role of SAH-induced apoptosis. 17b-Estradiol treatment may reverse apoptosis by increasing phospho-Akt and ERa protein expression in the dentate gyrus via an ERadependent pathway. Because E2 treatment holds therapeutic promise in the prevention of apoptosis after SAH, its role in patients and the mechanism for this change in specific areas merit further investigation.

Acknowledgment This work was supported by the National Science Council, ROC, under grants NSC 97-2314-B-037-012-MY2 and Chi-May 97CM-KMU-06.

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

references [21] [1] Cook DA. Mechanisms of cerebral vasospasm in subarachnoid haemorrhage. Pharmacol Ther 1995;66:259. [2] Nau R, Haase S, Bunkowski S, et al. Neuronal apoptosis in the dentate gyrus in humans with subarachnoid hemorrhage and cerebral hypoxia. Brain Pathol 2002;12:329. [3] Cahill J, Calvert JW, Zhang JH. Mechanisms of early brain injury after subarachnoid hemorrhage. J Cereb Blood Flow Metab 2006;26:1341. [4] Schievink WI, Riedinger M, Jhutty TK, et al. Racial disparities in subarachnoid hemorrhage mortality: Los Angeles County, California, 1985-1998. Neuroepidemiology 2004;23:299. [5] Lin CL, Dumont AS, Wu SC, et al. 17beta-estradiol inhibits endothelin-1 production and attenuates cerebral vasospasm after experimental subarachnoid hemorrhage. Exp Biol Med (Maywood) 2006;231:1054. [6] Lin CL, Shih HC, Dumont AS, et al. The effect of 17betaestradiol in attenuating experimental subarachnoid

[22]

[23]

[24]

[25]

[26]

e29

hemorrhage-induced cerebral vasospasm. J Neurosurg 2006; 104:298. Shih HC, Lin CL, Lee TY, et al. 17beta-Estradiol inhibits subarachnoid hemorrhage-induced inducible nitric oxide synthase gene expression by interfering with the nuclear factor kappa B transactivation. Stroke 2006;37:3025. Lin CL, Dumont AS, Su YF, et al. Attenuation of subarachnoid hemorrhage-induced apoptotic cell death with 17 betaestradiol: laboratory investigation. J Neurosurg 2009;111: 1014. Lin CL, Dumont AS, Su YF, et al. Attenuation of cerebral vasospasm and secondary injury by 17beta-estradiol following experimental subarachnoid hemorrhage. J Neurosurg 2009;110:457. Lin CL, Dumont AS, Tsai YJ, et al. 17Beta-estradiol activates adenosine A(2a) receptor after subarachnoid hemorrhage. J Surg Res 2009;157:208. Lin CL, Shih HC, Lieu AS, et al. Attenuation of experimental subarachnoid hemorrhageeinduced cerebral vasospasm by the adenosine A2A receptor agonist CGS 21680. J Neurosurg 2007;106:436. Kongable GL, Lanzino G, Germanson TP, et al. Gender-related differences in aneurysmal subarachnoid hemorrhage. J Neurosurg 1996;84:43. Wise PM, Dubal DB, Wilson ME, et al. Estrogens: trophic and protective factors in the adult brain. Front Neuroendocrinol 2001;22:33. Dubal DB, Kashon ML, Pettigrew LC, et al. Estradiol protects against ischemic injury. J Cereb Blood Flow Metab 1998;18: 1253. Schmidt R, Fazekas F, Reinhart B, et al. Estrogen replacement therapy in older women: a neuropsychological and brain MRI study. J Am Geriatr Soc 1996;44:1307. Tang MX, Maestre G, Tsai WY, et al. Effect of age, ethnicity, and head injury on the association between APOE genotypes and Alzheimer’s disease. Ann N Y Acad Sci 1996;802:6. Asselin E, Mills GB, Tsang BK. XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells. Cancer Res 2001;61:1862. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997;91:231. Panayotou G, Waterfield MD. The assembly of signalling complexes by receptor tyrosine kinases. BioEssays 1993;15: 171. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999;96:857. Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 1998; 282:1318. del Peso L, Gonzalez-Garcia M, Page C, et al. Interleukin-3induced phosphorylation of BAD through the protein kinase Akt. Science 1997;278:687. Cross DA, Alessi DR, Cohen P, et al. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995;378:785. Hetman M, Cavanaugh JE, Kimelman D, et al. Role of glycogen synthase kinase-3beta in neuronal apoptosis induced by trophic withdrawal. J Neurosci 2000;20:2567. Kennedy SG, Wagner AJ, Conzen SD, et al. The PI 3-kinase/ Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev 1997;11:701. Endo H, Nito C, Kamada H, et al. 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.

e30

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) e 2 3 ee 3 0

[27] Endo H, Nito C, Kamada H, et al. Akt/GSK3beta survival signaling is involved in acute brain injury after subarachnoid hemorrhage in rats. Stroke 2006;37:2140. [28] Noshita N, Lewen A, Sugawara T, et al. Akt phosphorylation and neuronal survival after traumatic brain injury in mice. Neurobiol Dis 2002;9:294. [29] Noshita N, Lewen A, Sugawara T, et al. Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice. J Cereb Blood Flow Metab 2001;21:1442. [30] Yu F, Sugawara T, Maier CM, et al. Akt/Bad signaling and motor neuron survival after spinal cord injury. Neurobiol Dise 2005;20:491. [31] Davis AM, Ellersieck MR, Grimm KM, et al. The effects of the selective estrogen receptor modulators, methyl-piperidinopyrazole (MPP), and raloxifene in normal and cancerous endometrial cell lines and in the murine uterus. Mol Reprod Dev 2006;73:1034. [32] Gingerich S, Krukoff TL. Estrogen in the paraventricular nucleus attenuates L-glutamate-induced increases in mean arterial pressure through estrogen receptor beta and NO. Hypertension 2006;48:1130. [33] Bonita R, Thomson S. Subarachnoid hemorrhage: epidemiology, diagnosis, management, and outcome. Stroke 1985;16:591. [34] Bederson JB, Levy AL, Ding WH, et al. Acute vasoconstriction after subarachnoid hemorrhage. Neurosurgery 1998;42:352. [35] Cahill J, Calvert JW, Marcantonio S, et al. p53 may play an orchestrating role in apoptotic cell death after experimental subarachnoid hemorrhage. Neurosurgery 2007;60:531. [36] Matz PG, Copin JC, Chan PH. Cell death after exposure to subarachnoid hemolysate correlates inversely with expression of CuZn-superoxide dismutase. Stroke 2000;31:2450. [37] Matz PG, Fujimura M, Chan PH. Subarachnoid hemolysate produces DNA fragmentation in a pattern similar to apoptosis in mouse brain. Brain Res 2000;858:312. [38] Park S, Yamaguchi M, Zhou C, et al. Neurovascular protection reduces early brain injury after subarachnoid hemorrhage. Stroke 2004;35:2412. [39] Prunell GF, Svendgaard NA, Alkass K, et al. Delayed cell death related to acute cerebral blood flow changes following subarachnoid hemorrhage in the rat brain. J Neurosurg 2005; 102:1046. [40] Zhou C, Yamaguchi M, Colohan AR, et al. Role of p53 and apoptosis in cerebral vasospasm after experimental subarachnoid hemorrhage. J Cereb Blood Flow Metab 2005; 25:572. [41] Yano S, Morioka M, Fukunaga K, et al. 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. [42] Cheng G, Chunlei W, Pei W, et al. Simvastatin activates Akt/glycogen synthase kinase-3beta signal and inhibits caspase-3 activation after experimental subarachnoid hemorrhage. Vascul Pharmacol 2010;52:77. [43] Endo H, Nito C, Kamada H, et al. Reduction in oxidative stress by superoxide dismutase overexpression attenuates acute brain injury after subarachnoid hemorrhage via activation of Akt/glycogen synthase kinase-3beta survival signaling. J Cereb Blood Flow Metab 2007;27:975. [44] Sugawara T, Ayer R, Jadhav V, et al. Simvastatin attenuation of cerebral vasospasm after subarachnoid hemorrhage in rats via increased phosphorylation of Akt and endothelial nitric oxide synthase. J Neurosci Res 2008;86:3635. [45] Santhanam AV, Smith LA, Akiyama M, et al. Role of endothelial NO synthase phosphorylation in cerebrovascular

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

protective effect of recombinant erythropoietin during subarachnoid hemorrhage-induced cerebral vasospasm. Stroke 2005;36:2731. Lee SY, Andoh T, Murphy DL, et al. 17beta-estradiol activates ICI 182,780-sensitive estrogen receptors and cyclic GMPdependent thioredoxin expression for neuroprotection. FASEB J 2003;17:947. Yang SH, He Z, Wu SS, et al. 17-beta estradiol can reduce secondary ischemic damage and mortality of subarachnoid hemorrhage. J Cereb Blood Flow Metab 2001;21:174. Liu Z, Gastard M, Verina T, et al. Estrogens modulate experimentally induced apoptosis of granule cells in the adult hippocampus. J Comp Neurol 2001;441:1. Nunes ML, Liptakova S, Veliskova J, et al. Malnutrition increases dentate granule cell proliferation in immature rats after status epilepticus. Epilepsia 2000;41(Suppl 6):S48. Culmsee C, Vedder H, Ravati A, et al. Neuroprotection by estrogens in a mouse model of focal cerebral ischemia and in cultured neurons: evidence for a receptor-independent antioxidative mechanism. J Cereb Blood Flow Metab 1999;19: 1263. Green PS, Simpkins JW. Neuroprotective effects of estrogens: potential mechanisms of action. Int J Dev Neurosci 2000;18: 347. Jover T, Tanaka H, Calderone A, et al. Estrogen protects against global ischemia-induced neuronal death and prevents activation of apoptotic signaling cascades in the hippocampal CA1. J Neurosci 2002;22:2115. Miller NR, Jover T, Cohen HW, et al. Estrogen can act via estrogen receptor alpha and beta to protect hippocampal neurons against global ischemia-induced cell death. Endocrinology 2005;146:3070. Ostrowski RP, Colohan AR, Zhang JH. Mechanisms of hyperbaric oxygen-induced neuroprotection in a rat model of subarachnoid hemorrhage. J Cereb Blood Flow Metab 2005; 25:554. Ferreri NR. Estrogen-TNF interactions and vascular inflammation. Am J Physiol Heart Circ Physiol 2007;292: H2566. Srivastava S, Weitzmann MN, Cenci S, et al. Estrogen decreases TNF gene expression by blocking JNK activity and the resulting production of c-Jun and JunD. J Clin Invest 1999; 104:503. Xing D, Feng W, Miller AP, et al. Estrogen modulates TNFalpha-induced inflammatory responses in rat aortic smooth muscle cells through estrogen receptor-beta activation. Am J Physiol Heart Circ Physiol 2007;292:H2607. Andersson C, Lydrup ML, Ferno M, et al. Immunocytochemical demonstration of oestrogen receptor beta in blood vessels of the female rat. J Endocrinol 2001;169:241. Darblade B, Pendaries C, Krust A, et al. Estradiol alters nitric oxide production in the mouse aorta through the alpha-, but not beta-, estrogen receptor. Circ Res 2002;90:413. Brann DW, Dhandapani K, Wakade C, et al. Neurotrophic and neuroprotective actions of estrogen: basic mechanisms and clinical implications. Steroids 2007;72:381. Kelleher RJ 3rd, Govindarajan A, Jung HY, et al. Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell 2004;116:467. Cabodi S, Moro L, Baj G, et al. p130Cas interacts with estrogen receptor alpha and modulates non-genomic estrogen signaling in breast cancer cells. J Cell Sci 2004;117: 1603. Koh PO, Cho JH, Won CK, et al. Estradiol attenuates the focal cerebral ischemic injury through mTOR/p70S6 kinase signaling pathway. Neurosci Lett 2008;436:62.