reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway

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Ischemic postconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway Yajing Yuan, Qulian Guo⁎, Zhi Ye, Xia Pingping, Na Wang, Zongbin Song Department of Anesthesiology, Xiangya Hospital of Central South University, Hunan 410008, China

A R T I C LE I N FO

AB S T R A C T

Article history:

Endoplasmic reticulum (ER) stress has been implicated in the pathology of cerebral

Accepted 5 October 2010

ischemia. During prolonged period of stress or when the adaptive response fails, apoptotic

Available online 1 November 2010

cell death ensues. Cerebral ischemic postconditioning (Postcond) has been shown to reduce cerebral ischemia/reperfusion (I/R) injury in both focal and global cerebral ischemia model.

Keywords:

However, the mechanism remains to be understood. This study aimed to elucidate whether

Ischemic postconditioning

Postcond attenuates brain I/R damage by suppressing ER stress-induced apoptosis and if the

Endoplasmic reticulum stress

phosphatidylinositol-3kinase/Akt (PI3K/Akt) pathway is involved. A focal cerebral ischemia

PI3K/Akt

rat model was used in the study. Rat brain infarct size and terminal deoxynucleotidyl

Apoptosis

transferase-mediated dUTP nick end labeling (TUNEL) positive cells in ischemic penumbra were assessed after reperfusion of the brain. The expressions of C/EBP-homologous protein (CHOP), caspase-12, glucose-regulated protein 78 (GRP78) and the phosphorylation of Akt (Ser473) in ischemic penumbra were measured after reperfusion. Our results showed that Postcond significantly attenuated brain I/R injury, as shown by reduction in infarct size, cell apoptosis, CHOP expression, caspase-12 activation and increase in GRP78 expression. LY294002, a phosphoinositide 3-kinase inhibitor, increased the number of TUNEL-positive cells suppressed by Postcond in penumbra. In addition, LY294002 diminished the effect of Postcond on the activation of CHOP, caspase-12 and GRP78. These results suggest that Postcond protects brain from I/R injury by suppressing ER stress-induced apoptosis and PI3K/Akt pathway is involved. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Following cerebral ischemia/reperfusion (I/R), neuron apoptosis occurs (Mattson et al., 2001). Although there are several pathways leading to cell apoptosis, the mitochondrial apoptotic pathway has been extensively studied (Eldadah and Faden, 2000; Fujimura et al., 1998). Both mitochondria and other organelles, such as the endoplasmic reticulum (ER), are

involved in intrinsic pathway of apoptosis (Deiss et al., 1996; Ferri and Kroemer, 2001; Mancini et al., 2000; Nakamura et al., 2000). A recent study of endoplasmic reticulum (ER) stress in regulating apoptosis in rat brain indicated that ER stressinduced apoptosis played an important role in the pathophysiology of neurodegeneration and cerebral ischemia (Arduino et al., 2009; Hayashi et al., 2003; Nakka et al., 2010; Chen and Gao, 2002). The proteins such as transcription factor C/

⁎ Corresponding author. Fax: +86 731 84327413. E-mail address: [email protected] (Q. Guo). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.10.017

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Fig. 1 – Effects of Postcond on infarct size. (A) Infarct size was detected by 2,3,5-triphenyl-2 H-tetrazolium chloride (TTC) staining 24 h after stroke. The columns show representative TTC staining from rat brains that received ischemia or Postcond with DMSO (dimethylsulfoxide) or with the PI3K inhibitor LY294002. (B) Postcond reduced infarct size in the brain section, whereas pretreatment of LY294002 partly eliminated the neuroprotection induced by Postcond. Data are expressed as mean ± S.D. *P < 0.05 vs. I/R group. #P < 0.05 vs. Postcond + DMSO group.

EBP homologous protein (CHOP) and caspase-12 were involved in ER stress-induced apoptosis (Zinszner et al., 1998; Szegezdi et al., 2006). The pro-apoptotic role of CHOP and caspase-12 in ER stress was confirmed in many studies (McCullough et al., 2001; Zinszner et al., 1998; Nakagawa and Yuan, 2000; Nakagawa et al., 2000). GRP78 is a main ER molecular chaperone and has its protective role in ER stress-mediated apoptosis both in vivo (Hayashi et al., 2003) and in vitro studies (Kishi et al., 2010).

Ischemic postconditioning is defined as a series of rapid intermittent interruptions of blood flow in the early phase of reperfusion that mechanically alters the hydrodynamics of reperfusion (Zhao et al., 2003b). In a previous study, Postcond protected the brain from I/R injury by inhibiting expression of apoptotic molecules in the mitochondrial pathway and by activating endogenous protective molecules (Xing et al., 2008). Liu et al. (2008) recently suggested that ischemic postconditioning is involved in suppression of I/R-induced ER stress. Thus, we speculate that neuroprotection induced by Postcond may be associated with ER stress-mediated apoptosis reduction. The mechanism of Postcond-induced neuroprotection through inhibition of ER stress-mediated apoptosis remains to be elucidated. PI3K/Akt pathway plays an important role in signal transduction pathways including cell growth, survival and metabolism. Gao et al. (2008) reported that phosphatidylinositol-3 kinase/Akt (PI3K/Akt) is an important factor in postconditioning induced neuroprotection. Akt counteracts endoplasmic reticulum stress-induced cell death (Hu et al., 2004) by reducing endoplasmic reticulum Ca2+ release (Marchi et al., 2008), and suppresses the activity of caspase-12 and CHOP expression (Shimoke et al., 2004; Hyoda et al., 2006). These findings suggest that Akt activation is a pro-survival pathway activated during ER stress, and PI3K/Akt pathway may be involved in suppression of ER stress-induced apoptosis. The purpose of our study is to investigate whether Postcond protects focal cerebral I/R injury through inhibiting ER stress-mediated apoptosis and whether PI3K/Akt pathway mediate the suppression of ER stress induced by Postcond.

2.

Results

2.1.

The effect of Postcond on infarct size in rat brain

The infarct size of brain in I/R group rats was 50.16 ± 3.34%. Ischemic Postcond reduced infarct size by approximately 29% (50.16 ± 3.34% vs. 21.08 ± 2.42%) in rats subjected to brain ischemia (Fig. 1). LY294002, given 15 min before Postcond, increased infarct size by 38% in rats with Postcond (Fig. 1). However, LY294002 did not completely reverse the protective effect of Postcond, because the infarct size in LY294002 + Postcond group rats was smaller than that in I/R group rats (38.50 ± 2.42% vs. 50.16 ± 3.34%) (Fig. 1). There were no significant differences in physiologic parameters (pH, pCO2 , pO2) in the experimental groups (see supplemental Table 1).

Fig. 2 – Effects of Postcond on expression of ER stress proteins CHOP, caspase-12, and GRP78 in the penumbra cortex of different group at different times of reperfusion. (A–C) Immunohistochemistries for CHOP(A), caspase-12(B), and GRP78(C) in the penumbra cortex at different times of reperfusion. Immunohistochemistries for CHOP and caspase-12 were negative and GRP78 were slight immunoreactivity in the sham group. In the I/R group, the immunoreactivities for CHOP, caspase-12, and GRP78 became gradually strong, and was strongest at 24 h of reperfusion. In the Postcond group, the immunoreactivities for CHOP and caspase-12 became gradually increased from 6 h to 24 h, but only to a milder degree. However, the immunoreactivity for GRP78 became much stronger from 12 h to 24 h of reperfusion. (D) Western blots analysis for CHOP, caspase-12, and GRP78 in the penumbra cortex of different group at different times of reperfusion. (E) Quantitative analysis of Western blotting showed that Postcond significantly decreased the expressions of CHOP and cleaved-caspase-12 at 24 h of reperfusion, but increased GRP78 expression from12 h to 24 h of reperfusion. Data are expressed as mean ± S.D. *P < 0.05 compared with I/R group at the same time points. Scale bar = 100 μm.

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2.2.

Effect of Postcond on the expression of ER stress proteins

CHOP, caspase-12 and GRP78 are good markers of ER stress, because they are expressed specifically under the conditions

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of ER dysfunction. In the present study, CHOP, caspase-12 and GRP78 in the penumbra cortex with immunohistochemical analysis and Western blot analysis at 6 h, 12 h and 24 h of reperfusion (Fig. 2). Immunoreactivities for CHOP and caspase-

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12 staining were negative in sham rats, while GRP78 were weakly detectable (Fig. 2A–C). In the I/R group, immunoreactivities for CHOP, caspase-12 and GRP78 in the penumbra cortex had gradually increased from 6 h to 24 h and peaked at 24 h of reperfusion. In comparison to the I/R group, the positive cells of both CHOP and caspase-12 significantly reduced in the Postcond group rats at 24 h of reperfusion. While the positive cells of GRP78 were significantly increased in the Postcond group rats from 12 h to 24 h of reperfusion (Fig. 2A–C). Western blot analysis showed small expressions of CHOP, cleaved-caspase-12 and GRP78 in the penumbra cortex in sham group rats (Fig. 2D). The amount of CHOP, cleavedcaspase-12 and GRP78 markedly increased after cerebral ischemia, especially at 24 h of reperfusion (Fig. 2E). As shown in (Fig. 2E), the expressions of CHOP and cleaved-caspase-12 in the Postcond group were significantly less than the I/R group at 24 h of reperfusion (P < 0.05). Postcond up-regulated GRP78 protein level from 12 h to 24 h of reperfusion (P < 0.05).

2.3. Postcond suppression of ER stress is mediated by PI3K/ Akt pathway In addition to its antiapoptotic effect, Postcond was shown to modulate signaling pathways (Gao et al., 2008). Postcond markedly increased P-Akt protein levels in the penumbra cortex at 24 h of reperfusion when compared with the sham and I/R groups (Fig. 3B–C). Compared with the sham group, the amount of total Akt decreased after stroke in the penumbra cortex. Pretreatment with PI3K inhibitor, LY294002, reduced PAkt increase (P < 0.05 vs. Postcond group) (Fig. 3C). We found that Postcond significantly decreased the expression of ER stress proteins at 24 h of reperfusion (Fig. 2E). So we further studied whether Postcond suppression of ER stress is mediated by PI3K/Akt pathway. We measured the changes in CHOP, caspase-12 and GRP78 with immunohistochemy and Western blot at 24 h of reperfusion (Fig. 3). Similar to the results as shown in Fig. 2, the expressions of CHOP and caspase-12 were decreased by Postcond, while GRP78 expression was increased by Postcond (Fig. 3). LY294002 treatment significantly increased the number of the positive cells of both CHOP and caspase-12 in the penumbra cortex at 24 h of reperfusion. However, the CHOP and caspese-12 positive neurons were more than that in the Postcond + DMSO group and less than that in the I/R group. Compared with the Postcond + DMSO group, GRP78-positive cells significantly decreased with LY294002 treatment (Fig. 3A). As shown in (Fig. 3B–C), Western blot analysis showed that PI3K inhibitor, LY294002, given 15 min before Postcond, abolished the Postcond effects on CHOP, cleaved-caspase-12 and GRP78 expressions (P < 0.05 vs. Postcond + DMSO group).

2.4.

Effect of Postcond on TUNEL staining

The result of TUNEL staining is shown in Fig. 4. TUNEL staining in the penumbra cortex was barely detectable in the sham group. TUNEL-positive cells in the penumbra cortex were slight positive at 6 h of reperfusion in I/R group rats, and were markedly increased from 12 h of reperfusion, then were strong positive at 24 h of reperfusion in the I/R group rats (Fig. 4A). The number of TUNEL-positive cells in the penumbra cortex

significantly reduced in the Postcond group, compared with the I/R group at 24 h of reperfusion (Fig. 4B). However, LY294002 increased the number of TUNEL-positive cells (Fig. 4C–D).

3.

Discussion

Postcond provided neuroprotective effect in rats after focal cerebral ischemia (Taskapilioglu et al., 2009; Xing et al., 2008; Zhao et al., 2006a; Pignataro et al., 2008). However, its mechanisms are still not well understood. Our study focused on the neuroprotective mechanism of Postcond. In our study, Postcond significantly decreased infarct size in rat brain after 30 min of brain ischemia followed by 24 h of reperfusion. Postcond protect against cerebral I/R injury by reducing ER stress-induced apoptosis. We measured the brain CHOP, caspase-12, and GRP78 level at 6 h, 12 h and 24 h of reperfusion. Our results showed that Postcond inhibited the expression of CHOP and caspase-12 activation , but it increased the expression of GRP78. Postcond also reduced the number of apoptotic cells in the penumbra cortex after reperfusion. These effects were mediated by the PI3K/Akt signaling pathway. The molecular mechanism of Postcond on neuroprotection is not fully elucidated yet. A previous study demonstrated that Postcond inhibits focal cerebral I/R injury by antiapoptotic mechanisms (Xing et al., 2008). The neuroprotective effect was mediated through inhibiting apoptosis molecules of the mitochondrial pathway (Xing et al., 2008). Recently, endoplasmic reticulum (ER) stress was found to be involved in regulating apoptosis (Hayashi et al., 2003; Chen and Gao, 2002). Cerebral ischemia causes severe ER stress that results in ER function disruption and unfolded proteins accumulation in the ER lumen, ultimately leads to cell death (Hu et al., 2000; Tajiri et al., 2004). There was a significant reduction in the number of TUNEL-positive cells in the penumbra cortex in the Postcond group rats at 24 h of reperfusion in our study. To investigate if Postcond reduced apoptosis induced by I/R injury and if this effect is related to ER stress, we next examined the expression of CHOP, caspase-12, and GRP78. Brain I/R injury up-regulates the expression of ER stress markers such as CHOP, caspase-12 and GRP78 (Nakka et al., 2010; Kudo et al., 2008) that results in ER stress-associated apoptosis (Hayashi et al., 2003). Similar to their studies, our results also demonstrated that I/R injury increases CHOP expression, caspase-12 activation, GRP78 induction and apoptosis in the penumbra cortex after cerebral ischemia. The proapoptotic transcription factor CHOP plays a key role in ER stress-induced apoptosis (Ma et al., 2002; Maytin et al., 2001; Wang et al., 1996). Down-regulation of CHOP activity compromises cell viability, and cells lacking CHOP are significantly protected from the lethal ER stress (Zinszner et al., 1998). Our results showed that Postcond significantly decreased the activation of CHOP at 24 h of reperfusion. CHOP sensitizes cells to ER stress induced-apoptosis through down-regulation of the anti-apoptotic factor B cell lymphoma-2 (Bcl-2) and upregulation of reactive oxygen species (ROS) (Marciniak et al., 2004; McCullough et al., 2001). Thus, we hypothesize that some molecules of CHOP pathway, such as Bcl-2 and ROS, may contribute to the decreased TUNEL-positive cells by Postcond.

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Fig. 3 – Postcond suppression of ER stress is mediated by PI3K/Akt pathway. (A) Immunohistochemistries for CHOP, caspase-12, and GRP78 in the penumbra cortex at 24 h of reperfusion. Immunohistochemical reactions for CHOP, caspase-12 were negative and GRP78 were slight immunoreactivity in the sham group. In the I/R group, the immunohistochemical reactions for CHOP, caspase-12 and GRP78 became much strong. However, in the Postcond + DMSO group, the immunohistochemistry for CHOP, caspase-12 decreased to a milder degree. But the immunohistochemistry for GRP78 further increased and became much stronger. With LY294002 treatment, the immunohistochemistry for CHOP and caspase-12 increased to strong immunoreactivity, and the immunohistochemistry for GRP78 returned to mild degree. (B) Western blots analysis for CHOP, caspase-12, and GRP78 in the penumbra cortex of different group at 24 h of reperfusion. (C) Quantitative analysis showed that Postcond suppressed the activation of CHOP and caspase-12 and increased the induction of GRP78 and P-Akt. However, pretreatment of LY294002 partly eliminated the Postcond effects on the expression of CHOP, caspase-12, and GRP78. Data are expressed as mean ± S.D. *P < 0.05 vs. I/R group. #P < 0.05 vs. Postcond + DMSO group. Scale bar = 100 μm.

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Fig. 4 – Effect of Postcond on neuronal apoptosis after cerebral ischemia. (A) TUNEL staining was performed on the sections from ischemic penumbra. Magnification is 400×. (B) The statistical analysis of apoptotic neurons in the ischemic penumbra of different group at different times of reperfusion. Postcond significantly reduced the number of TUNEL-positive cells at 24 h of reperfusion in the ischemic penumbra. (C–D) However, pretreatment of LY294002 (LY) partly eliminated the anti-apoptosis effect induced by Postcond. Data are expressed as mean ± S.D. *P < 0.05 vs. I/R group. #P < 0.05 vs. Postcond + DMSO group.

Caspase-12, a murine protein associated with the ER membrane, normally exists in an inactive procaspase form (Nakagawa et al., 2000). During ER stress, caspase-12 dissociates from the ER membrane and is cleaved to a fragment, then it is activated. Once it is activated, caspase-12 initiates downstream apoptotic pathways (Nakagawa et al., 2000). Caspase-12-deficient mice are resistant to ER stress-induced apoptosis (Nakagawa and Yuan, 2000). Our results showed that the activation of caspase-12 was significantly suppressed by Postcond at 24 h of reperfusion. GRP78, a main ER molecular chaperone, regulates protein folding and facilitates protein translocation in the ER and protein secretion (Liang and MacRae, 1997). The up-regulation of GRP78 leads to proper protein folding, and its protective roles were demonstrated in an in vitro study that GRP78-depleted cells exhibited increase susceptibility to tunicamycin treatment and excitotoxicity (Yu et al., 1999). In agreement with the finding of Hayashi et al. (2003), our study showed that Postcond up-regulated the expression of GRP78 in the penumbra cortex. Increased expression of GRP78 attenuated the induction of CHOP during ER stress and reduced ER stress-induced apoptosis in one study (Wang et al., 1996). Thus, we believe that increased expression of GRP78 contributes to Postcond-induced neuroprotective effect against cerebral ischemia. Our study suggests that Postcond targets and reduces ER stress induced-apoptosis. The signaling pathway by which Postcond regulates ER stress-associated apoptosis remains unclear. The Akt signaling pathway contributes to postconditioning's protection against

stroke (Gao et al., 2008; Zhao et al., 2006b; Pignataro et al., 2008). Postconditioning significantly increased Akt kinase activity at 5 h post-stroke (Gao et al., 2008). We tested if PI3K/Akt pathway is involved in Postcond-induced suppression of ER stressmediated apoptosis at 24 h of reperfusion following brain ischemia. Our results showed that Postcond increased Akt phosphorylation and a PI3K inhibitor increased brain infarct size. PI3K/Akt pathway mediating ER stress-induced cell death was reported (Hu et al., 2004). Activation of the PI3K/AKT pathway was a positive regulator of ER stress-induced apoptosis (Hu et al., 2004; Srinivasan et al., 2005). In our present study, LY294002 was used to confirm whether PI3K/Akt pathway is involved in the Postcond-induced suppression of ER stressmediated apoptosis. The PI3K inhibitor abolished the protective effects of Postcond on ER stress-induced apoptosis. LY294002 not only decreased GRP78 expression but also increased CHOP induction, caspase-12 activation and TUNEL-positive cells in the penumbra cortex. A recent study (Morishima et al., 2004) showed that translocation of Bim to the ER during stress is an important mechanism by which caspase-12 is activated and ER stress-induced apoptosis starts. More studies are needed to determine whether the activation of PI3K/Akt pathway induced by Postcond contributes the reduction of translocation of Bim to the ER. In conclusion, Postcond protects focal cerebral I/R injury through inhibiting ER stress-mediated apoptosis, and PI3K/Akt pathway is involved in the Postcond-induced suppression of ER stress-mediated apoptosis.

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4.

Experimental procedures

4.1.

Animals

immersion in 4% paraformaldehyde. The percentage of infarct cortex was measured by normalizing to the entire ipsilateral cortex from animals as described previously (Zhao et al., 2003a).

4.5. Adult male Sprague–Dawley (SD) rats weighing 350–450 g were housed under diurnal lighting conditions (12 h dark/light). Experiments were performed according to the international guidelines for animal research. All experiments were approved by the Institutional Animal Care and Use Committee of Legacy Research. Animals were randomly divided into five groups for the experiment: (1) Sham group (n=14) : rats underwent the same surgical procedure without ischemia. (2) I/R group (n = 48): ischemia for 30 min followed by 6 h, 12 h, and 24 h of reperfusion. (3) Postcond group (n=42): ischemia plus Postcond, then followed by 6 h, 12 h, and 24 h of reperfusion. (4) Postcond+DMSO group (n=20): Postcond with coinjection of 3% dimethyl sulfoxide only (10 μl, icv, 15 min before Postcond; Sigma, St. Louis, MO, USA) (Zhao et al., 2005), then followed by 24 h of reperfusion. (5) Postcond+LY294002 (Postcond + LY) group (n=20): Postcond with coinjection of LY294002 (10 μl, 10 mM, in 3% dimethyl sulfoxide, icv, 15 min before Postcond; Sigma, St. Louis, MO, USA) (Zhao et al., 2005), then followed by 24 h of reperfusion.

4.2.

Focal cerebral ischemia

Focal cerebral ischemia was induced as described previously (Zhao et al., 2003a, 2005). In brief, rats were anesthetized with 10% chloral hydrate (350 mg/kg, i.p.) and the rectal temperature was kept at 37 °C with a homoeothermic blanket during whole experiments. The left distal MCA was exposed and cauterized permanently above the rhinal fissure. The bilateral common carotid arteries (CCA) were occluded for 30 min with suture tightening and then released to allow partial reperfusion through collateral blood flow. Tail artery samples were taken and physiological parameters (pH, pCO2 and pO2) were measured using blood gas analyzer (International Technidyne Co., USA).

Western blotting analysis

Ischemia postconditioning

Ischemic postconditioning (Postcond) was performed as described previously by Zhao et al. (2006a). CCAs were occluded by aneurysms clips for 30 min after MCA occlusion and then released, while the distal MCA remained occluded permanently. After 30 s of CCA reperfusion, the CCAs were occluded again by tightening sutures around the CCA for 10 s, followed with another 2 cycles of 30 s reperfusion and 10 s occlusion (total of 3 cycles), and then allowed reperfusion for 6 h, 12 h and 24 h.

4.4.

Immunohistochemical study and TUNEL staining

Anesthetized animals (n = 6 at each time point) were perfused with 4% paraformaldehyde. Brains were removed and postfixed for 24 h with 4% paraformaldehyde, the coronal sections were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min and then embedded in paraffin. The embedded tissues were cut into 4 μm thickness, and incubated with primary antibodies in 10% normal goat serum and 0.3% Triton-X 100 overnight. The primary antibodies used were as follows: a 1:100 dilution of goat polyclonal anti-GRP78 antibody or rabbit polyclonal anti-CHOP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a 1:500 dilution of rabbit polyclonal anti-caspase-12 antibody (Abcam plc Cambridge, UK). Sections were treated simultaneously without the first antibodies as negative control. After quenching endogenous peroxidase activity by exposing slides to 0.3% H2O2 and 10% methanol for 20 min, slides were washed in PBS and incubated for 2 h with biotinylated secondary antibodies: a 1:200 dilution of anti- goat IgGs or anti- rabbit IgGs (Beijing Zhong Shan-Golden Bridge Biological Technology CO., LTD), subsequently, they were incubated with avidin–biotin–horseradish peroxidase complex. Sections were stained with DAB (Vector, CA, USA) /H2O2 solution for coloration. For TUNEL study, the sections (n = 6 at each time point) were treated according to the manufacturer's instructions (Roche Molecular Biochemicals, Inc., Mannheim, Germany). In brief, after pretreatment with Proteinase K and 0.3% H2O2, the sections were incubated with terminal deoxynucleotidyl transferase enzyme at 37 °C for 1 h. The sections then were incubated with peroxidase-conjugated antibody for 30 min at . 3′-Diaminobenzidine (DAB) was used for the coloration of apoptotic cells. The photographs were taken under highpower magnification (×400).

4.6. 4.3.

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Measurement of infarct volume

At 24 h after reperfusion, rats (n = 6 each group) were decapitated and the brains were rapidly removed. Infarct sizes were measured by 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC; Sigma, St. Louis, MO, USA) (Zhao et al., 2005). Brains were cut into 2-mm-thick coronal sections in a cutting block and stained with 1% TTC solution for 30 min at 37 °C followed by overnight

Ischemic penumbra cortical samples (n = 8 at each time point) were homogenized in a lysis buffer (50 mmol/L Tris–HCl, pH 7.5,100 mmol/L NaCl, 1% Triton X-100) containing protease inhibitors (aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and pepstatin), and phosphatase inhibitor (Sigma cocktail). After centrifugation at 10,000g at 4 °C for 5 min, the supernatants were collected. Protein concentration was estimated using the Badford reagent (Sigma, St Louis, MO, USA). Then, the protein was mixed with Laemmli sample buffer and heated at 99 °C for 5 min. For Western blot analysis an equal amount of protein (50–100 μg) was loaded in each well and subjected to 10–15% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were then transferred from the gel to polyvinylidinene fluoride (Millipore, Bedford, MA, USA) membranes and blocked in 5% non-fat dry milk prepared in 1× TBST. The membranes were incubated with the primary antibodies overnight at 4 °C. The following primary antibodies were used: GRP-78 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CHOP (1:200, Santa Cruz Biotechnology,

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Santa Cruz, CA, USA), caspase-12 (1:1000, Abcam plc Cambridge, UK), Akt (1:1000, Cell Signaling Technology, Beverly, MA, USA), PAkt (1:1000, Cell Signaling Technology, Beverly, MA, USA), or βactin antibody(1:1000, Abcam, Cambridge, MA, USA). After washing primary antibodies with 1× TBST, the membranes were incubated with appropriate secondary antibodies for 2 h at room temperature. The blots were developed using ECL (Beyotime Institute of Biotechnology) plus detection system and the relative band density was measured using FluorChem FC2 System (NatureGene Corp., USA).

4.7.

Statistical analysis

Data are expressed as mean ± S.D. Statistical analysis was performed with analysis of variance (ANOVA) followed by Student–Newman–Keuls or Dunnet's test (SPSS, Inc., Cary, NC, USA). P < 0.05 was considered statistically significant. Supplementary materials related to this article can be found online at doi:10.1016/j.brainres.2010.10.017.

Acknowledgments We are thankful to Fenghua Li, MD and Reza Gorji, MD for their help in writing this manuscript. Both Dr. Li and Dr. Gorji are faculty in the Department of Anesthesiology at the SUNY Upstate Medicial University, Syacuse, NY 13210, USA.

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