Akt pathway mediates geranylgeranylacetone-induced neuroprotection against cerebral infarction in rats

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Research Report

The phosphatidylinositol-3 kinase/Akt pathway mediates geranylgeranylacetone-induced neuroprotection against cerebral infarction in rats Eiji Abe, Minoru Fujiki⁎, Yasuyuki Nagai, Kong Shiqi, Takeshi Kubo, Keisuke Ishii, Tatsuya Abe, Hidenori Kobayashi Department of Neurosurgery, School of Medicine, Oita University, 1-1, Idaigaoka, Hasama-machi, Oita, 879-5593, Japan

A R T I C LE I N FO

AB S T R A C T

Article history:

Previous studies demonstrated the cytoprotective effect of geranylgeranylacetone (GGA), a heat

Accepted 22 February 2010

shock protein inducer, against ischemic insult. Phosphatidylinositol-3 kinase/Akt (PI3K/Akt) is

Available online 3 March 2010

thought to be an important factor that mediates neuroprotection. However, the signaling pathways in the brain in vivo after oral GGA administration remain unclear. We measured and

Keywords:

compared infarction volumes to investigate the effect of GGA on cerebral infarction induced by

Cerebral ischemia

permanent middle cerebral artery occlusion in rats. We evaluated the effects of pretreatment

Geranylgeranylacetone

with 5-hydroxydecanoate (5HD), a specific mitochondrial ATP-sensitive potassium (mitoKATP)

Specific mitochondrial

channel inhibitor; diazoxide (DZX), a selective mitoKATP channel opener and wortmannin

ATP-sensitive potassium channel

(Wort), a specific PI3K inhibitor of GGA-induced neuroprotection against infarction volumes. To

PI3K/Akt pathway

clarify the relationship between PI3K/Akt activation and neuroprotection, we used immunoblot

Neuroprotection

analysis to determine the amount of p-Akt proteins present after GGA administration with or without Wort treatment. Neuroprotective effects of GGA (pretreatment with a single oral GGA dose (800 mg/kg) 48 h before ischemia) were prevented by 5HD, DZX and Wort pretreatment, which indicates that the selective mitoKATP channel and the PI3K/Akt pathway may mediate GGA-dependent protection. Oral GGA-induced p-Akt and GGA pretreatment enhanced ischemia-induced p-Akt, both of which were prevented by Wort pretreatment. These results suggest that a single oral dose of GGA induces p-Akt and that GGA plays an important role in neuroprotection against cerebral ischemia through the mitoKATP channel opening. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Recent studies have demonstrated that a single oral dose of geranylgeranylacetone (GGA) is a convenient noninvasive pretreatment strategy for neuronal protection against ischemic insult (Fujiki et al., 2006, 2003; Nagai et al., 2005). However, the signaling pathways in the rat brain after GGA administration

remain to be defined. Antiapoptotic activity of Akt is mediated through activation of the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway (Gross, 2005). The mitochondrial ATPsensitive potassium (mitoKATP) channel was reported to be involved in cerebral (Watanabe et al., 2008; Mayanagi et al., 2007) and myocardial (Pell et al., 1997; Shinohara et al., 2007) ischemic preconditioning. There is strong support for the

⁎ Corresponding author. Fax: +81 97 586 5869. E-mail address: [email protected] (M. Fujiki). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.02.074

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hypothesis that mitoKATP channel openers activate the PI3K/ Akt pathway in vitro (Bijur and Jope, 2003). We postulated that the PI3K/Akt pathway is upstream of the mitoKATP channel, and that activation of the latter leads to preservation of neuroprotective and neurological functions. In this study, we used a rat experimental model for focal cerebral ischemia after permanent middle cerebral artery occlusion (MCAO) to investigate the neuroprotective role of GGA. Specifically, we tested the hypothesis that oral GGA would induce p-Akt and, thereafter, opening of the mitoKATP channel is involved in neuroprotection against cerebral ischemia. This is the first report that investigates the role of the PI3K/Akt pathway and the mitoKATP channel in the neuroprotective mechanism of GGA.

2.

Results

2.1.

Infarction volume and neurological score

No significant differences were found in body temperature and other physiological variables (blood pH, blood gases and hematocrit) between the groups. No differences in physiological variables were apparent between the groups with different GGA–ischemia intervals and those with different GGA concentrations (data not shown). GGA significantly reduced the volume of the infarction induced by MCAO in rats. As has been previously demonstrated,

the greatest degree of preservation of the cerebral hemisphere occurred after 800 mg/kg, but only when GGA was given 48 h before MCAO (Nagai et al., 2005). Animals pretreated with DZX alone also showed statistically significant reduced infarction volumes (P < 0.05; Fig. 1). In addition, the 800 mg/kg GGApretreated ischemia animals showed a statistically significantly reduced neurological deficit (control group: 8.50± 0.423; 5HD group: 8.875 ± 0.441; GGA–5HD group: 8.25± 0.62; DZX group: 12.125± 0.398; GGA–DZX group: 8.125 ± 0.441; GGA group: 13.875 ± 0.549; dimethyl sulfoxide (DMSO) control group: 8.25± 0.526; Wort group: 7.75 ± 0.25; Wort–GGA group: 8.375 ± 0.532; DMSO– GGA group: 13.75 ± 0.648; Table 1). There were statistically significant correlations between neurological scores and infarction volumes in the GGA group and the DZX group (P < 0.05). Coinjection of 5HD, DZX and Wort with GGA abolished the protective effects, but 5HD and Wort alone did not influence the protective effects with regard to the volume of surviving cerebral tissue (2,3,5-triphenyltetrazolium chloride (TTC)stained) 24 h after MCAO (Figs. 1 and 2). Animals pretreated with 5HD alone tended to show reduced infarction volumes, but this was not significant.

2.2.

Increases in p-Akt levels after GGA administration

One-way analysis of variance performed on p-Akt expression showed the primary effects of treatment [F(6, 21): 5.75, P < 0.005]. Specifically, GGA pretreatment enhanced ischemiainduced p-Akt expression. Pretreatment with Wort alone and

Fig. 1 – Effect of 5HD and DZX treatment combined with GGA on infarction volume after MCAO. Results are shown for animals given treatment with vehicle control (A), 5HD pretreatment (B), DZX pretreatment (C), GGA pretreatment (D), GGA pretreatment with coinjection of 5HD (E), GGA pretreatment with coinjection of DZX (F). Note the extensive cerebral infarction in the right hemisphere in the animals subjected to ischemia alone and the reduction of infarction area in the animals that received 800 mg/kg GGA as preconditioning. Coinjection of 5HD or DZX with GGA abolished the protective effects, but 5HD alone did not influence the protective effects in terms of the volume of surviving cerebral tissue (n = 6 in each group). The graphs indicate mean values, and the bars indicate SEM for infarction volume (G). *P < 0.05, vs. vehicle-treated control group with MCAO. + P < 0.05, vs. group treated with GGA and 5HD or GGA and DZX. NS, not significant.

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Table 1 – Neurological score 24 h after middle cerebral artery (MCA) occlusion.

GGA 5HD DZX Mean SEM

Con

5HD

DZX

GGA

GGA–5HD

GGA–DZX

– – – 8.5 0.423

– + – 8.875 0.441

– – + 12.125 a 0.398

+ – – 13.875 a 0.549

+ + – 8.25 0.62

+ – + 8.125 0.441

Con GGA Wort

Wort

GGA

Wort–GGA

– –

– +

+ –

+ +

8.25 0.526

7.75 0.25

13.75 b 0.648

8.375 0.532

n = 6 per groups. 5HD: 5-hydroxydecanoate, DZX: diazoxide, GGA: geranylgeranylacetone, Wort: Wortmannin. a p < 0.0001, compared with vehicle pretreated MCA occlusion control (0 mg/kg of GGA). b p < 0.0001, compared with DMSO and vehicle pretreated control (pretreatment with DMSO before vehicle administration).

with Wort before GGA administration 48 h before occlusion did not influence ischemia-induced p-Akt expression (Fig. 3). This resulted in no significant difference among the control, Wort– vehicle and Wort–GGA groups. p-Akt expression in animals without ischemia 3 h after administration increased in the GGA group compared with the control group (P < 0.05; Fig. 3). Pretreatment with Wort before GGA administration suppressed GGA-induced p-Akt expression.

Fig. 2 – Effect of Wort treatment combined with GGA on infarction volume after MCAO. The graph shows the average infarction volume for animals in the vehicle control group and the icv control group (vehicle administration alone and 5 μl in 100% DMSO icv 30 min before vehicle administration, respectively), the Wort pretreatment group and the GGA pretreatment group. Note the extensive cerebral infarction in the animals subjected to ischemia alone and the reduction of infarction area in the animals that received 800 mg/kg GGA as preconditioning. Coinjection of Wort with GGA abolished the protective effects, but Wort alone did not influence the protective effects in terms of the volume of surviving cerebral tissue (n = 6 in each group). The graphs indicate mean values, and the bars indicate SEM for infarction volume (G). *P < 0.05, vs. vehicle-treated control group with MCAO. +P < 0.05, vs. Wort-treated group. ++P < 0.05, vs. GGA + Wort-treated group. NS, not significant.

3.

Discussion

The present results demonstrate that GGA alone induces a great deal of p-Akt, GGA pretreatment enhances ischemiainduced p-Akt (as measured by immunoblotting), and Wort, a specific PI3K inhibitor, shows a statistically insignificant protective tendency after focal cerebral ischemia. Previous studies have revealed that after preconditioning, p-Akt peaks

Fig. 3 – Quantitative analyses of the increases in p-Akt levels after GGA administration as revealed by Western blot analysis. (A) Effects of Wort pretreatment on GGA-induced p-Akt expressions (3 h after administration) and ischemia-induced p-Akt expression 1 h after ischemia. Representative blots of p-Akt and t-Akt expressions as revealed by Western blot analysis in seven groups. (B) Quantitative evaluation of p-Akt expressions in seven groups revealed p-Akt increased after ischemia. GGA substantially enhanced the ischemia-induced p-Akt expression (n=4 in each group). The values represent the mean and SEM. *P<0.05, vs. vehicle-treated control group with MCAO. **P<0.01, vs. GGA-treated group with MCAO. NS, not significant.

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at 1–4 h after treatment and returns to normal values within 24 h (Li et al., 2008; Yin et al., 2007). The present results demonstrated that a single oral dose of GGA also leads to peak upregulation of p-Akt at 3 h post administration, which then returns to normal. The route of drug administration (e.g., intravenous) could affect the time course and outcome of the investigations. The amount of each drug that could pass through the blood–brain barrier and systemic drug metabolism could also play an important role and exert a direct effect on the infarction volume in this study. In this regard, it would be more interesting if we could try an alternative method of administration with methodological development of GGA for intravenous or intracerebral ventricle (icv) administration. Pretreatment with Wort before GGA administration substantially suppressed GGA-induced p-Akt expression. If GGAinduced neuroprotective effects depend only on p-Akt expression, the neuroprotective tendency should not be found in Wort–GGA animals. In line with the degree of infarction size and the level of neurological deficit, the level of Akt phosphorylation at 1 h after infarction was enhanced by GGA administration. The neuroprotective effects in the GGA group were completely counteracted by pretreatment with Wort, suggesting the PI3K/ Akt pathway plays an important role even though the role of Akt phosphorylation remains controversial. Studies have shown that ischemic preconditioning protects the brain by induction of ischemic tolerance resulting from a sublethal ischemic insult accompanied by PI3K/Akt pathway activation (Yano et al., 2001). The PI3K/Akt pathway is a central mediator in signal transduction pathways involved in cell growth, survival and metabolism. Akt phosphorylates caspase 9 at Ser-196, thereby blocking cytochrome c-mediated caspase 9 activation in vitro (Cardone et al., 1998). Akt may rescue cells from apoptosis by inhibiting the Bax-dependent apoptosis pathway through a forkhead box transcription factor (Nakae et al., 2000). It would be important to clarify whether GGA activates PI3K/ Akt signaling, thereby indirectly mediating the mitoKATP channel, or whether GGA mediates the mitoKATP channel directly. Although the association between Akt phosphorylation and the mitoKATP channel has not been well clarified, our observations may be explained by the hypothesis that Akt phosphorylation and subsequent opening of the mitoKATP channel play a part in the protective mechanisms when GGA-pretreated brains are subjected to lethal cerebral infarctions. The important role of the mitoKATP channel in cerebral ischemic tolerance after benign ischemic preconditioning has been reported (Watanabe et al., 2008; Mayanagi et al., 2007; Shimizu et al., 2002). Administrations of 20–40 mg/kg 5HD 24 h after benign ischemic preconditioning appropriately inhibited the mitoKATP channel (Watanabe et al., 2008; Mayanagi et al., 2007). In the present study, 40 mg/kg 5HD was used. This concentration completely eliminated the neuroprotective effect observed in the GGA group. This observation suggests that the neuroprotective effects afforded by GGA preconditioning predominantly depend on opening of the mitoKATP channel. To examine whether preconditioning with GGA is affected by experimental mitoKATP channel conditions, we added a subgroup pretreated with GGA and then treated with a selective mitoKATP channel opener, DZX. Unexpectedly, in contrast to our result using DZX alone 24 h before ischemia, DZX failed to elicit protection in animals pretreated with GGA 48 h before ischemia. The reason for this finding is unclear but may involve complex

interactions at the level of the mitochondria. Several actions of GGA, including activation of the mitoKATP channel and protein kinase C (PKC) activation, also occur with DZX (Kis et al., 2003). Activation of PKC modulates cell viability pathways, which either protects neuronal cells (Ding et al., 1997; Maher, 2001) or induces cell death (Datta et al., 1997; Majumder et al., 2000). PKC α and ε activate extracellular signal-regulated kinases (ERKs) and Jun Nterminal kinase, and inhibit p38 mitogen-activated protein kinase activation (Maher, 2001). PKC ε activity at the mitochondrial level may contribute to regulation of adenosine-induced mK+ATP channels, maintaining energy and reducing calcium influx during ischemia (Bright et al., 2004). In our previous study, the GGA-preconditioned animals tended to show moderate increases in PKC ε, even though this effect was not statistically significant; this result suggests that GGA-induced PKCε confers protection against cerebral ischemia, in part, by maintaining mitochondrial function via ERK activity and potentially by mediating the function of mK+ATP channels (Fujiki et al., 2006). In contrast, studies of the brain have demonstrated that PKC δ (as opposed to other PKC isoenzymes) is associated with increased neuronal death. Inhibition of PKC δ with a PKC δ-selective inhibitor peptide significantly reduced cerebral tissue damage when administered at the onset of reperfusion (Bright et al., 2004). Moreover, overexpression of PKC δ can induce or potentiate programmed cell death (Ghayur et al., 1996). Thus, PKC δ activation is not likely to explain directly GGA's neuroprotective, but neurotoxic at high concentrations, effect on cerebral infarction. Furthermore, a recent report demonstrated that chronic Akt activation preceding ischemia–reperfusion in the heart resulted in increased injury and decreased functional recovery after the event, which may account for our result that the mitoKATP channel opener DZX (activates PI3K/Akt pathway) abolished the protective effect of GGA-induced p-Akt expression (Nagoshi et al., 2005). Given the results obtained with the PI3K inhibitor Wort, our present results provide evidence to support the notion that Akt phosphorylation mediated the opening of mitoKATP channels, which are a downstream target of Akt. These are early steps in the development of GGA-induced preconditioning (Shinohara et al., 2007). It is possible that GGA's immediate activation effects on mitochondria and/or its preceding interactive activation effects on PKC and PI3K/Akt is able to negate the delayed effects of DZX by activating competing pathways. A reduction in neurobehavioral deficits tended to correspond with the degree of neuroprotection, probably because neurological function after ischemia depends not only on the degree of spared area but also on the degree of expression of factors such as neurotrophic factors, which might involve functional recovery and plasticity after ischemia (Currie et al., 2000). Thus, these results suggest that GGA-induced p-Akt protects against some, but not all, central nervous system injuries. GGA mediates and induces proteins such as PKC, Ras and thioredoxin (which may act as an antioxidant), as well as p-Akt, all of which inhibit cascades of apoptosis resulting from Bad, procaspase 9 and Bax-independent pathway inactivation (Barone et al., 1998; Yamanaka et al., 2003). It is not clear whether GGA would cause any of these conditions. Blocking of Akt activation by administering Wort before GGA blocked the neuroprotective action of preconditioning. Because Akt was activated after brain ischemia, the upstream signaling that underlies Akt activation in both sublethal ischemia and lethal

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ischemia is possibly associated with activation of Akt signaling during ischemia (Yano et al., 2001). The transcriptional activator CREB is phosphorylated by Akt both in vitro and in situ at Ser133 (Du and Montminy, 1998). The phosphorylation of Ser-133 increases binding of CREB to CBP and enhances CREB-mediated transcription (Du and Montminy, 1998). The relevance of CREB phosphorylation by Akt in the survival signaling is still unclear, although there is evidence that CREB-regulates expression of genes critical for survival such as those encoding cytokines and BDNF (Tao et al., 1998). Hence, if neuroprotective genes are directly induced by GGA-induced p-AKt, the factors that mediate the induction remain to be defined. Any of these alterations in gene expression could help protect against cerebral infarction. p-Akt or other neuroprotective genes may promote neuron survival or mediate neuroprotection, but how they are induced by GGA and how the protective genes actually operate remain to be established.

4.

Experimental procedures

4.1.

Animals and experimental protocol

All experimental protocols were approved by the Oita University Ethical Review Committee. Male Wistar rats (body weight 290–375 g) were housed at controlled room temperature (24.5 °C–25.0 °C) with a 12/12-h light/dark cycle. The rats had free access to food pellets and tap water. GGA, as an emulsion with 5% gum arabic and 0.008% tocopherol, was given orally before MCA. The oral dose of 800 mg/kg was based on previous observations (Nagai et al., 2005) showing that a single oral GGA dose (800 mg/kg) 48 h before ischemia significantly attenuated cerebral infarction volume. In one experiment, the correlation with neuroprotection against MCAO was examined by coinjecting 5HD (40 mg/kg, ip,

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24 h before MCAO; Sigma, St. Louis, MO, USA), a specific mitochondrial ATP-sensitive potassium channel inhibitor; diazoxide (DZX; 10 mg/kg, ip, 24 h before MCAO; Sigma, St. Louis, MO, USA), a selective mitochondrial ATP-sensitive potassium channel opener (Mayanagi et al., 2007; Lenzser et al., 2005; Yamanaka et al., 2003) and wortmannin (Wort; 16 µg/kg, 5 µl in 100% DMSO, via a 5-µl Hamilton syringe into the icv, 30 min before 800 mg/kg GGA; Sigma, St. Louis, MO, USA), a specific PI3K inhibitor (Li et al., 2008; Yin et al., 2007). Animals were classified into six groups for the first experiment: a control group (vehicle administration, n=6); a 5HD group (vehicle administration with coinjection of 5HD, n= 6); a GGA group (800 mg/kg GGA pretreatment, n= 6); a GGA–5HD group (GGA pretreatment with coinjection of 5HD, n =6); a DZX group (vehicle administration with coinjection of DZX, n =6) and a GGA– DZX group (GGA pretreatment with coinjection of DZX, n=6). Animals were classified into five groups for the second experiment: a control group (vehicle administration alone and 5 µl in 100% DMSO icv 30 min before vehicle administration, n = 6 each); a Wort group (vehicle administration with coinjection of Wort, n = 6); a GGA group (800 mg/kg GGA pretreatment, n = 6) and a Wort–GGA group (GGA pretreatment with coinjection of Wort, n = 6). On the basis of our hypothesis that the PI3K/Akt pathway is upstream of the mitoKATP channel, and that activation of the latter leads to neuroprotection, Wort and DMSO were applied before GGA administration, and 5HD and DZX were applied after GGA administration. Furthermore, data from the group treated with Wort were separated from the results of groups treated with other drugs, because icv administration of Wort or DMSO 30 min before GGA administration was a fundamentally different manipulation than that used in the 5HD, DZX and GGA groups. Ischemia was induced 48 h after the oral administration of GGA or vehicle. GGA dose and GGA–ischemia interval have been chosen on the basis of our previous work (Nagai et al., 2005; Fig. 4).

Fig. 4 – Experimental protocol. Control group: vehicle administration before MCAO (n = 6). 5HD group: vehicle administration with coinjection of 5HD (n = 6). DZX group: vehicle administration with coinjection of DZX (n = 6). GGA group: 800 mg/kg GGA pretreatment (n = 6). GGA–5HD group: GGA pretreatment with coinjection of 5HD (n = 6). GGA–DZX group: GGA pretreatment with coinjection of DZX (n = 6). Second control group: vehicle administration alone and 5 μl in 100% DMSO icv 30 min before vehicle administration (n = 10). Wort group: vehicle administration with coinjection of Wort (n = 10). GGA group: 800 mg/kg GGA pretreatment with coinjection of DMSO (n = 10). Wort–GGA group: GGA pretreatment with coinjection of Wort (n = 10). Animals were sacrificed 3 h after oral administration (n = 4 in each group) or 1 h after onset of ischemia (n = 4 in each group) for p-Akt and t-Akt Western blot analysis. Administration route: icv, intracerebral ventricle; ip, intraperitoneal; oral.

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Correlations with p-Akt induction after GGA administration with or without ischemia were examined by coinjecting Wort (16 µg/kg, icv, 30 min before 800 mg/kg GGA or vehicle). In this experiment, animals with ischemia were classified into four groups: a control group (pretreatment with DMSO before vehicle administration, n = 10); a GGA group (800 mg/kg GGA pretreatment, n = 10); a Wort–vehicle group (pretreatment with Wort before vehicle administration, n = 10) and a Wort–GGA group (pretreatment with Wort before GGA administration, n = 10). Animals without ischemia were classified into three groups: a control group (pretreatment with DMSO before vehicle administration, n = 4); a GGA group (800 mg/kg GGA pretreatment, n = 4) and a Wort–GGA group (pretreatment with Wort before GGA administration, n = 4). For Western blot analysis, the animals were sacrificed 3 h after oral administration (n = 4 in each group) or 1 h after onset of ischemia induced 48 h after administration (n = 4 in each group). We accepted the time point 3 h after oral administration after reviewing data of the preliminary experiment (2 h, 3 h, 4 h after oral administration; data not shown).

4.2.

Middle cerebral artery occlusion model

The animals were anesthetized with pentobarbital (40 mg/kg ip). We used a temperature-controlled heating pad to maintain body temperature at 37 °C throughout surgery and during the postsurgical recovery period. Physiological variables (pH, pCO2, pO2 and hematocrit) were measured in 0.1 mL of arterial blood from a right femoral catheter with the use of a blood analysis system (International Technidyne Co., USA). Focal cerebral ischemia was achieved by the permanent intraluminal suture occlusion method described by Longa et al (1989). Briefly, under an operating microscope, the left common, external and internal carotid arteries were identified, and carefully dissected free from surrounding nerves and fascia through a ventral cervical midline incision. The common carotid artery (CCA) and external carotid artery were isolated, carefully separated from the adjacent vagus nerve and ligated with a 6-0 nylon suture. A 25-mm length of 3-0 nylon suture was aseptically introduced into the internal carotid artery (ICA) from the CCA, approximately 2 mm proximal to the bifurcation through a puncture. The suture was secured with a 6-0 nylon ligature at an ICA origin and gently advanced exactly 18 mm intracranially from the bifurcation of the CCA to embed into the left anterior cerebral artery so that the left MCA was occluded at its origin. The neck incision was closed with silk sutures. During the experiments, laser Doppler flow (LDF) was monitored with an LDF meter (TBF-LN1; Unique Medical, Tokyo, Japan) attached to the intact skull, positioned 1 mm posterior and 3 mm lateral to the bregma, and overlying the MCA territory.

4.3.

Measurement of infarction volume

Twenty-four hours after MCAO, brains were cut into 7 coronal slices 2 mm thick. These slices were reacted with a 2% solution of TTC to reveal the ischemic infarction. Unstained areas of infarction of the left cerebral hemisphere were then traced and measured by a computerized image analysis system (NIH Image; National Institutes of Health, Bethesda, MD, USA), and

the infarction areas were calculated to obtain the infarction volume (mm3) per brain.

4.4.

Evaluation of neurological deficit

Neurological deficits at 24 h after ischemia were examined with the scoring scale described by Garcia et al (1995). Specifically, we evaluated spontaneous activity, symmetry in the movement of four limbs, forepaw outstretching, climbing, body proprioception and response to vibrissae touch. These six tests were each scored from 0 to 3, which mean that behavioral deficits were graded by a total score of 0 to 18. All behavioral evaluations were made by an observer blinded to the treatment group. Neurological score and infarction volume of animals were evaluated in double-blind fashion without knowledge of the treatment group.

4.5.

Western blot analysis for Akt

For Western blot analysis, the right hemisphere was transferred to an ice-cold mammalian tissue lysis/extraction reagent (Sigma, St. Louis, MO, USA) that contained a protease inhibitor cocktail (Sigma). The tissues were then homogenized. The homogenate was centrifuged at 1500 × g for 10 min at 4 °C. The supernatant was stored at − 80 °C. Protein concentrations were determined by a previously described method (Yamanaka et al., 2003) with bovine serum albumin used as a standard (Bio-Rad). Equivalents of 40-μg protein extracts from brain tissue were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein separation was performed in 10% polyacrylamide with 0.05% bisacrylamide. The proteins were then electrophoretically transferred to polyvinylidene difluoride membranes. After being transferred and blocked with 0.5% nonfat milk, the membranes were incubated with polyclonal antibodies (rabbit anti-Akt, 1:400, and mouse anti-phospho-Akt, 1:400; Cell Signaling). Signals were detected by electrochemiluminescence with exposure to Hyperfilm (Amersham Pharmacia). Each blot was probed for total Akt as an internal control to ensure equivalent protein loading and protein integrity. The amount of protein on the immunoblots was quantified with NIH Image software.

4.6.

Statistical analysis

All data are represented as mean ± standard error of the mean (SEM). Different groups of animals were compared by using the two-tailed Student t-test for independent pairs or oneway (two-way for time course) ANOVA with the Student– Newman–Keul post hoc analysis (SPSS, Inc., Cary, NC, USA). Differences were considered significant at the level of probability P ≤ 0.05.

Acknowledgments This work was supported by a grant from the Japan Science and Technology Agency and the Ministry of Education, Culture, Sports, Science and Technology. The authors are

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grateful to Y. Hagimori for her technical assistance and to Eisai Co. Ltd. for the gift of GGA.

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