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Akt pathway
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Research Report
Remote ischemic postconditioning protects the brain from global cerebral ischemia/reperfusion injury by up-regulating endothelial nitric oxide synthase through the PI3K/Akt pathway Bei Penga , Qu-lian Guoa,⁎, Zhi-jing Heb , Zhi Yea , Ya-jing Yuana , Na Wanga , Jun Zhouc a
Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, PR China Department of Oral and Maxillofacial Surgery, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, PR China c Center of Experimental Medicine, The Third Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, PR China b
A R T I C LE I N FO
AB S T R A C T
Article history:
Remote ischemic postconditioning (RIPoC) attenuates ischemia/reperfusion (I/R) injury in the
Accepted 16 January 2012
heart, lung and hind limb. RIPoC performed in the hind limb reduces brain injury following
Available online 26 January 2012
focal cerebral ischemia in rats. Whether RIPoC has a neuroprotective effect with respect to global cerebral I/R injury is, however, unknown, and the mechanism of neuroprotection
Keywords:
needs further elucidation. Here we investigated whether RIPoC could reduce global cerebral
Global cerebral ischemia
I/R injury in rats and whether this neuroprotective effect was induced by up-regulating endo-
Remote ischemic postconditioning
thelial nitric oxide synthase (eNOS) through the phosphatidylinositol-3 kinase/Akt (PI3K/Akt)
Delayed neuronal death
pathway. Global cerebral ischemia was performed via 8 min of four-vessel occlusion. Neuronal
Phosphatidylinositol-3 kinase/Akt
density, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-
Endothelial nitric oxide synthase
positive cells and expression of Bcl-2 and Bax in the hippocampal CA1 region were assessed after reperfusion. Morris water maze task was used to quantify spatial learning and memory deficits after reperfusion. The expression of eNOS, phosphorylated eNOS (Ser1177), Akt and phosphorylated Akt (Ser473) in the CA1 region was measured after reperfusion. RIPoC significantly attenuated delayed neuronal death and reduced the spatial learning and memory deficits associated with global cerebral ischemia. Pre-administration of N(ω)-nitro-L-arginine methyl ester (a nonselective NOS inhibitor) significantly abolished the neuroprotective effect of RIPoC. Moreover, pre-administration of LY294002 (a highly selective inhibitor of PI3K) not only significantly reversed the neuroprotective effect of RIPoC, but also obviously inhibited the up-regulation of eNOS induced by RIPoC. Our findings suggest that RIPoC protects the brain against global cerebral I/R injury and that this neuroprotection is mediated by upregulating eNOS through the PI3K/Akt pathway. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: + 86 731 84327413. E-mail address: [email protected] (Q. Guo). Abbreviations: 4-VO, four-vessel occlusion; ANOVA, analysis of variance; CCAs, common carotid arteries; DMSO, dimethyl sulfoxide; eNOS, endothelial nitric oxide synthase; I/R, ischemia and reperfusion; iNOS, inducible nitric oxide synthase; L-NAME, N(ω)-nitro-L-arginine methyl ester; LY, LY294002; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; PI3K, phosphoinositide-3 kinase; RIPoC, remote ischemic postconditioning; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling 0006-8993/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.01.033
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1.
Introduction
Global cerebral ischemia can develop in clinical scenarios that deprive the brain of oxygen and glucose for a short period of time, including carotid endarterectomy, cardiac arrest and cardiopulmonary bypass surgery (Lee et al., 2000; Liang et al., 2008). Rapid reperfusion is the most effective treatment for ischemia, minimizing both structural and functional injuries. Paradoxically, however, restoration of cerebral blood flow causes further damage to the ischemic brain (Frantseva et al., 2001; Tsubota et al., 2010). Many studies have attempted to identify pharmacological or physical interventions that effectively protect the brain against cerebral ischemia and reperfusion (I/R) injury, both global and focal. Ischemic postconditioning, which refers to a short period of sub-lethal ischemia, limits I/R injury in both myocardial and cerebral infarctions when performed immediately or shortly after reperfusion (Chao et al., 2010; Sandu and Schaller, 2010; Wang et al., 2008; Xing et al., 2008). A fascinating new intervention, termed remote ischemic postconditioning (RIPoC), has recently emerged as an effective way to ameliorate I/R injury in the heart, lung and hind limb (Gritsopoulos et al., 2009; Kerendi et al., 2005; Tsubota et al., 2010). Ren et al. (2009) first reported that RIPoC performed in the ipsilateral hind limb can reduce the cerebral infarct size following focal cerebral ischemia. It is, however, not known whether RIPoC can reduce I/R injury following global cerebral ischemia, which has a different pathophysiology than does focal cerebral ischemia. Although several molecular mechanisms are involved in the protective effect of RIPoC against I/R injury in other organs (Andreka et al., 2007; Gritsopoulos et al., 2009; Kerendi et al., 2005), the mechanism of neuroprotection afforded by RIPoC against cerebral I/R injury has not been fully elucidated. Nitric oxide synthase (NOS) plays an important role in physiological and pathological events in the central nervous system. Endothelial NOS (eNOS) is found in a subset of neurons and in the endothelium of cerebral blood vessels. Nitric oxide (NO) generated by eNOS is crucial for vascular function and homeostasis and plays a critical role in the protection of ischemic preconditioning (Chen et al., 2010a, 2010b; Liu et al., 2006; Scorziello et al., 2007) and ischemic postconditioning (Liu et al., 2007) against I/R injury. Using L-arginine or physical activity to up-regulate eNOS increases cerebral blood flow and protects against cerebral ischemia (Lin et al., 2010). The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in signal transduction related to cell growth, proliferation, differentiation, motility, survival and metabolism (Hui et al., 2005). In addition, it is also an important factor in the neuroprotective effect of ischemic postconditioning, as it regulates the downstream anti-apoptotic and survival molecules (Gao et al., 2008). Akt, which is activated by phosphorylation via activated PI3K, phosphorylates eNOS on serine 1177 (p-eNOS), thereby activating this enzyme (Endres et al., 2004; Hashiguchi et al., 2004). Therefore, we speculated that upregulation of eNOS by PI3K/Akt may account for the acquisition of a neuroprotective effect during global cerebral I/R injury following RIPoC. In this study, we investigated whether RIPoC can protect the brain against global cerebral I/R injury in rats and whether
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the neuroprotective effect is associated with up-regulation of eNOS mediated by the PI3K/Akt pathway.
2.
Results
2.1. RIPoC protected against global cerebral ischemia/reperfusion injury RIPoC in the I/R + RIPoC group significantly attenuated global cerebral I/R injury as compared with the I/R group (Figs. 1–2). As shown by Nissl staining (Figs. 1A–B), no significant differences in neuronal density were observed between the sham and RIPoC groups (P > 0.05). In contrast, neuronal density was significantly decreased in the I/R group as compared with the sham group (Fig. 1B). The number of intact neurons in the I/R + RIPoC group was significantly greater than that in the I/R group (Fig. 1B). In the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, TUNEL-positive cells in the CA1 region were analyzed after 24 h and 48 h of reperfusion (Figs. 1C–D). Apoptotic cells were infrequently detected in both the sham and RIPoC groups (Fig. 1D). Compared with the sham group, numerous TUNEL-positive cells were detected in the I/R group (Fig. 1D). RIPoC significantly reversed the increase in TUNEL-positive cells in the I/R + RIPoC group as compared with the I/R group (Fig. 1D). Western blot analysis was used to quantify the expression of apoptosis-related proteins Bcl-2 and Bax in the CA1 region after 24 h and 48 h of reperfusion (Figs. 1E–G). Four-vessel occlusion (4-VO) significantly affected the expression of both Bcl-2 and Bax (Figs. 1F–G). Compared with the I/R group, RIPoC significantly up-regulated the levels of Bcl-2 (Fig. 1F) and significantly down-regulated Bax levels (Fig. 1G) in the I/R + RIPoC group. To investigate whether postconditioning strategies that reduced neuronal death also improved behavioral deficits, we next tested the spatial learning and memory of rats in the Morris water maze task (Fig. 2). During the training phase, significant differences in mean escape latency between both training days [F(3,84) = 200.600, P < 0.01] and between groups [F(3,28) = 27.830, P < 0.01] were detected. In addition, an interaction between the training day and group factors was detected [F(9,84) = 4.280, P < 0.01]. Compared with rats from the sham and RIPoC groups, the I/R group took longer to find the hidden platform on all training days (Fig. 2A). The prolonged escape latency of the I/R group was significantly reduced by RIPoC (Fig. 2A). Concerning spatial learning ability, no significant difference between the sham and RIPoC groups was detected at any stage of the training (P > 0.05; Fig. 2A). During the probing phase, the I/R group made fewer platform crossings than did either the sham or RIPoC group, and RIPoC treatment (in the I/R + RIPoC group) significantly increased the number of crossings over the platform site as compared with the I/R group (Fig. 2B). Differences between the sham and RIPoC groups were not significant (P > 0.05). Finally, the percentage of time spent in the target quadrant was used to estimate retention performance (Fig. 2C). The I/R group spent less time in the target quadrant as compared with either the sham or RIPoC
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Fig. 1 – RIPoC attenuated delayed neuronal death and inhibited neuronal apoptosis in the hippocampal CA1 region induced by global cerebral I/R. (A) Representative Nissl staining of the CA1 neurons in the sham, I/R, I/R+ RIPoC and RIPoC groups after 7 days of reperfusion (400×). (B) Neuronal density of surviving neurons in the CA1 region (n= 8). (C) Representative TUNEL staining of the CA1 neurons in the sham, I/R, I/R + RIPoC and RIPoC groups (400×) after 24 h and 48 h of reperfusion. (D) The number of TUNEL-positive cells in the CA1 region in each group (n = 6). (E) Representative blots of Bcl-2 and Bax of the CA1 region in the sham, I/R and I/R + RIPoC groups after 24 h and 48 h of reperfusion (n = 6). (F, G) Relative protein levels of Bcl-2 and Bax based on band densities of Western blots using β-actin as a control. Data are expressed as mean ± SD. Scale bar: 50 μm. *: P < 0.01 vs. the sham group; #: P < 0.01 vs. the I/R group; @: P < 0.01 vs. the I/R + RIPoC group.
group. A significant difference was observed between the I/R and I/R + RIPoC groups, whereas no significant differences were detected between the I/R + RIPoC and RIPoC groups (P > 0.05, Fig. 2C).
2.2. Inhibition of NOS or PI3K reduced the neuroprotective effect of RIPoC To test whether NOS and the PI3K/Akt pathway are crucial to RIPoC-mediated reduction of delayed neuronal death, we
used pharmacological interventions and pre-treated rats with N(ω)-nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) or LY294002 (LY, a highly selective inhibitor of PI3K) (Fig. 3). Neuronal density was significantly decreased in L-NAME + I/R + RIPoC and LY + I/R + RIPoC groups as compared with the I/R + RIPoC group (Fig. 3B). L-NAME and LY significantly reversed the decrease in the number of TUNEL-positive cells that was observed following RIPoC (Fig. 3D). In the Morris water maze task, pre-administration of L-NAME or LY significantly increased the escape latency
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Fig. 2 – RIPoC reduced spatial learning and memory deficits induced by global cerebral I/R. The Morris water maze was performed to test the spatial learning and memory in the sham, I/R, I/R+ RIPoC and RIPoC groups from 4th to 7th day after reperfusion. (A) Escape latency to find the hidden platform during four consecutive days of training. (B) The number of times the rats crossed over the former platform location during the probe trial. (C) The percent of time spent in the target quadrant during the probe trial. (D) Representative swimming paths during the probe trial for each group. Data are expressed as mean ± SD (n = 8). *: P < 0.01 vs. the sham group; #: P < 0.01 vs. the I/R group.
(Fig. 3E), decreased the number of crossings over the platform site (Fig. 3F) and reduced the percentage of time spent in the target quadrant as compared with values for the I/R + RIPoC group (Fig. 3G).
up-regulated expression of p-eNOS and eNOS after reperfusion (Figs. 4B–C).
3. 2.3. RIPoC induced the up-regulation of eNOS that was mediated by the PI3K/Akt pathway To investigate whether RIPoC induced up-regulation of eNOS in the CA1 region and whether this change was mediated through the PI3K/Akt pathway, we carried out the RIPoC procedure in the presence and absence of L-NAME or LY and then measured the protein levels of p-eNOS, eNOS, p-Akt and Akt in the CA1 region after 48 h of reperfusion (Fig. 4). RIPoC significantly increased the expressions of p-eNOS, eNOS and p-Akt when compared with levels in the sham and I/R groups (Figs. 4B–D). L-NAME significantly reversed the increase in p-eNOS and eNOS that was induced by RIPoC following 4-VO as compared with amounts in the I/R + RIPoC group (Figs. 4B–C). Moreover, LY not only significantly prevented the activation of p-Akt that was induced by RIPoC following 4-VO (Fig. 4D), but also significantly reversed the
Discussion
In the present study, 3 cycles of 15-min occlusion/15-min release of the bilateral femoral arteries immediately after 4-VO was selected as the RIPoC protocol. This approach was based on the previous research of Ren et al. (2009), who first expanded the concept of conventional ischemic postconditioning to include remote ischemic postconditioning, which was induced in a distant non-vital organ, in a rat model of focal cerebral I/R. The present study described and investigated the neuroprotective role of RIPoC after global cerebral ischemia via 4-VO. These results demonstrated that RIPoC attenuated delayed neuronal death and spatial learning and memory deficits induced by global cerebral I/R. This neuroprotective effect is apparently involved in regulating the apoptotic proteins Bcl-2 and Bax. Furthermore, inhibition of NOS with L-NAME abolished the attenuation of global cerebral I/R injury provided by RIPoC. Pre-treatment with LY not only prevented the neuroprotective
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Fig. 4 – RIPoC induced the up-regulation of eNOS in the hippocampal CA1 region and this up-regulation was mediated by the PI3K/Akt pathway. Representative Western blot (A) and quantitative analysis of p-eNOS (B), eNOS (C), p-Akt (D) and Akt (E) expressions of the CA1 region of rats in the sham, I/R, I/R + RIPoC, L-NAME + I/R + RIPoC, and LY + I/R + RIPoC groups after 48 h of reperfusion. L-NAME reversed the RIPoC-induced increase in p-eNOS and eNOS following 4-VO as compared with the I/R + RIPoC group. Moreover, LY not only significantly prevented the activation of p-Akt that was induced by RIPoC following 4-VO, but also significantly reversed the up-regulated expression of p-eNOS and eNOS after reperfusion. Data are expressed as mean ± SD (n = 6). *: P < 0.01 vs. the sham group; #: P < 0.01 vs. the I/R group; @: P < 0.01 vs. the I/R + RIPoC group.
effect of RIPoC, but also inhibited RIPoC-induced up-regulation of eNOS in the CA1 region after reperfusion. The brain is intrinsically vulnerable to global ischemia, especially in the CA1 region of the hippocampus (Connell et al., 2011; Lee et al., 2000). In our experiments, RIPoC effectively inhibited neuronal cell apoptosis after 24 and 48 h of reperfusion and reduced delayed neuronal death after 7 days of reperfusion in the CA1 region. Global cerebral ischemia, which leads to the most extensive neuronal damage in the CA1 region, results in a deficit in spatial learning and memory (Wang et al., 2008). Thus, the Morris water maze task was used to quantify spatial learning and memory deficits of rats subjected to global cerebral ischemia. Four-VO significantly impaired spatial learning and memory. RIPoC decreased the
escape latency, increased the number of platform site crossings, and increased the time spent in the target quadrant. It seems, therefore, that neurons rescued by the RIPoC treatment maintained at least some of their functional parameters. Our results are consistent with those of Ren et al. (2009), which showed that RIPoC is protective in the rat model of focal cerebral ischemia (middle cerebral artery occlusion), an occurrence that has a pathophysiology that is very different from that of global cerebral ischemia (Hashiguchi et al., 2004). NO and its derivatives can have multiple effects that affect neuronal death in different ways. It should be mentioned that the roles of NO and NOS during I/R are controversial (Brown, 2010). NO from NOS has both neurotoxic and neuroprotective roles during cerebral I/R (Endres et al., 2004; Scorziello et al.,
Fig. 3 – Inhibition of NOS or PI3K reversed the neuroprotective effects of RIPoC. (A) Representative Nissl staining of the hippocampal CA1 neurons in the I/R, I/R + RIPoC, L-NAME + I/R + RIPoC and LY + I/R + RIPoC groups after 7 days of reperfusion (400×). (B) Neuronal density of surviving neurons in the CA1 region (n = 8). (C) Representative TUNEL staining of the CA1 neurons in the I/R, I/R + RIPoC, L-NAME+ I/R +RIPoC and LY + I/R + RIPoC groups (400×) after 48 h of reperfusion. (D) The number of TUNEL-positive cells in the CA1 region (n= 6). (E, F, G, H) The spatial learning and memory were tested in the I/R, I/R + RIPoC, L-NAME + I/R + RIPoC and LY + I/R+ RIPoC groups from 4th to 7th day after reperfusion (n= 8). (E) Escape latency to find the hidden platform during four consecutive days of training. (F) The number of times the rats crossed over the former platform location during the probe trial. (G) The percent of time spent in the target quadrant during the probe trial. (H) Representative swimming paths during the probe trial for each group. Data are expressed as mean± SD. Scale bar: 50 μm. #: P < 0.01 vs. the I/R group; @: P < 0.01 vs. the I/R + RIPoC group; $: P < 0.05 vs. the I/R + RIPoC group.
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2007). Excessive production of NO during the early minutes of reperfusion can produce I/R-induced injury via lipid peroxidation, DNA damage and pro-apoptotic effects (Liu et al., 2007; Zhao et al., 2010). In contrast, NO is protective based on its ability to cause vasodilatation, inhibit platelet plug formation and reduce the inflammatory response, as well as its anti-apoptotic actions (Chen et al., 2010a, 2010b; Zhao et al., 2007). NO can induce protection via cyclic guanosine monophosphate-mediated vasodilation and mitochondrial permeability transition inhibition by reactive oxygen and nitrogen species-mediated activation of the NF-κB and mitogen-activated protein kinase pathways or via S-nitros(yl)ation of caspases or the N-methyl-D-aspartate receptor (Brown, 2010; Hashiguchi et al., 2004). The cellular effects of NO may be dependent on its concentration, site of release and duration of action (Liu et al., 2009; Rastaldo et al., 2007). We showed that L-NAME, a nonselective NOS inhibitor, abolished the neuroprotective effect induced by RIPoC following 4-VO. This strongly suggests that, in the case of the neuroprotective effect of RIPoC against global cerebral I/R injury, the neuroprotective role of NO and NOS is more relevant than their neurotoxic role. The three isoforms of NOS play different roles in ischemic brain damage: eNOS protects (probably by maintaining vasodilation and promoting angiogenesis), neuronal NOS (nNOS) mediates neuronal damage (probably by leading to peroxynitrite production) and inducible NOS (iNOS) can play protective or damaging roles or both (Hashiguchi et al., 2004). nNOS knockout mice were partially protected against ischemia-induced neuronal death (Brown, 2010). Three different strains of iNOS knockout showed no effect of iNOS on infarct size after transient focal brain ischemia, and there was no induction of iNOS expression by ischemia (Pruss et al., 2008). NO induced by eNOS can also protect neurons against focal ischemia in the brain via the vasodilatory action of NO (Hashiguchi et al., 2004). Therefore, we focused on the expression of eNOS after reperfusion instead of iNOS and nNOS in the present study. NO produced by eNOS is important in vascular signaling and tone regulation and leukocyte–endothelial interactions (Lin et al., 2010). eNOS-deficient mice are hypertensive and have an increased infarct volume after a stroke (Endres et al., 2004; Sawada and Liao, 2009). In contrast, transgenic mice in which eNOS is overexpressed are resistant to stress-induced hypotension, inflammation and stroke (Yamashita et al., 2000). Up-regulation of eNOS in neurons and endothelium was reported to be protective, and the mechanism that leads to this effect may involve anti-apoptosis (De Palma et al., 2008). In the current study, RIPoC significantly up-regulated the expression of eNOS and p-eNOS in the CA1 region after global cerebral I/R, and inhibition of NOS significantly reversed the neuroprotective effect of RIPoC on global cerebral I/R injury. These results suggest that eNOS and NO are involved in the neuroprotective effect of RIPoC in the rat model of global cerebral ischemia. L-NAME is an arginine analog commonly used as a potent and reversible L-arginine competitive inhibitor of NOS. Nevertheless, it was demonstrated that L-NAME reduced the up-regulation of eNOS and p-eNOS expressions induced by RIPoC in the CA1 region. And previous studies showed that pretreatment with L-NAME inhibited the limb ischemic preconditioning induced increase of eNOS activities in the hippocampus and serum in global cerebral I/R injury (Zhao et al., 2007), reduced the upregulated expressions of eNOS and p-eNOS by eNOS gene
transfer in myocardial infarction (Chen et al., 2007, 2010a, 2010b), decreased the increased level of eNOS in liver by ischemic postconditioning in liver warm I/R injury (Guo et al., 2011) and abolished the induction of the expression of eNOS by Ginsenoside Rb1 in myocardial I/R injury (Xia et al., 2011). However, the mechanism remains unclearly understood. We speculated that L-NAME intraperitoneally may reduce the expression and activation of eNOS in the CA1 region by some other signaling pathways during the reperfusion of the brain in this study. Therefore, future studies need to address why L-NAME reduced RIPoC-induced eNOS expression and phosphorylation in the CA1 region. Among the several signaling pathways that up-regulate eNOS following various pharmacological stimuli, we were particularly interested in the PI3K/Akt pathway. The PI3K/Akt pathway is involved in the neuroprotective effect of ischemic postconditioning (Yuan et al., 2011; Zhou et al., 2011). In the rat model of global cerebral ischemia in this study, RIPoC increased Akt phosphorylation, and the PI3K inhibitor LY, which prevents the phosphorylation and activation of Akt, significantly inhibited RIPoC-induced neuroprotection. Akt phosphorylates eNOS at Ser1177 in neurons and endothelial cells, thereby activating the enzyme (Hashiguchi et al., 2004; Lin et al., 2010). The activation of eNOS by Akt might contribute to the important physiological effects on apoptosis, cell attachment and cell proliferation (Dimmeler et al., 1999). In our study, inhibition of the PI3K/Akt pathway significantly prevented the RIPoC-induced increase of eNOS and p-eNOS expressions in the CA1 region after reperfusion. In addition, the PI3K/Akt pathway-mediated up-regulation of eNOS is required for the anti-apoptotic effect of insulin, estrogen and statins in stroke (Endres et al., 2004). PI3K/Akt signaling leading to eNOS activation in neurons and endothelial cells contributes to ischemic tolerance in the rat hippocampus (Lin et al., 2010). The data in this study suggest that the activation of the PI3K/Akt pathway by protective stimuli in the hind limbs not only was involved in the neuroprotective effect of RIPoC, but also mediated the up-regulation of eNOS in the CA1 region that was induced by RIPoC. In the present study, we assessed whether RIPoC treatment alone has an effect on delayed neuronal death and spatial learning and memory. Our data show that RIPoC had an insignificant effect on neuronal density, the TUNEL assay results and the Morris water maze task if 4-VO had not been previously carried out. We therefore have not assessed the effect of RIPoC treatment alone on the expression of apoptosisrelated proteins Bcl-2 and Bax in the CA1 region. Furthermore, although vehicle-only treatments were not included in the investigation of the potential role and relationship of the PI3K/Akt pathway and eNOS in the neuroprotective effect of RIPoC, a single injection of normal saline or dimethyl sulfoxide (DMSO) has been reported to have no effect on the NOS or the PI3K/Akt pathway (Yuan et al., 2011; Zhou et al., 2011). In fact, DMSO does not affect PI3K and is commonly used as a solvent for pharmacological agents (Liu et al., 2010). Therefore, we believe that L-NAME and LY, and not their associated solvents, reversed the RIPoC-mediated neuroprotective effects. RIPoC is a simple and harmless method that provides a new tool to prevent I/R injury. Shown clearly were the feasibility and safety in application of RIPoC as an adjunct to current
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reperfusion strategies. It would be practical to create limb ischemia by placing a tourniquet on an extremity for a brief period of time and then releasing it after cerebral reperfusion (Li et al., 2006; Ren et al., 2009). In conclusion, repetitive brief sub-lethal I/R in the hind limbs may result in the activation of molecular pathways responsible for RIPoC, thereby protecting the brain from global cerebral I/R injury. RIPoC appeared to attenuate delayed neuronal death and ameliorate spatial learning and memory deficits that resulted from global cerebral ischemia. This neuroprotective effect was mediated by up-regulating eNOS through the PI3K/Akt pathway.
4.
Experimental procedures
4.1.
Animals
Adult male Sprague–Dawley rats (200–250 g), obtained from the Experimental Animal Centre of Central South University, were used for this study according to the Guide for the Care and Use of Laboratory Animals (Zimmermann, 1983). The animal experiments were approved by the animal ethics committee of Xiangya Hospital Central South University. All the rats were housed in cages in a temperature-controlled (24 ± 1 °C) room and were maintained under diurnal lighting conditions (12 h light/dark). They had free access to food and water. Pain and suffering to the rats were minimized. Prior to the experiments, rats were fed in the laboratory animal room for 2 days to allow for adaptation to the environment. 4.2.
Experimental protocols
To investigate the neuroprotective effects of RIPoC against global cerebral I/R injury, rats were randomly divided to four groups: (1) the sham group (n = 20): rats were subjected to sham operations without 4-VO and RIPoC. (2) The I/R group (n = 32): rats were subjected to 4-VO followed by 24 h, 48 h and 7 days of reperfusion and sham RIPoC. (3) The I/R + RIPoC group (n = 32): rats were subjected to 4-VO followed by RIPoC and 24 h, 48 h and 7 days of reperfusion. (4) The RIPoC group (n = 32): rats were subjected to sham 4-VO followed by RIPoC and 24 h, 48 h and 7 days of reperfusion. TUNEL assay and Western blot analysis were performed after 24 h and 48 h of reperfusion. The spatial learning and memory were tested in Morris water maze task from the 4th to 7th day after reperfusion. Delayed neuronal death in the hippocampal CA1 region was examined by Nissl staining after 7 days of reperfusion. To investigate the potential roles and relationship of PI3K/Akt pathway and eNOS in neuroprotection of RIPoC, rats were randomly divided to additional two groups: (5) the L-NAME + I/R + RIPoC group (n = 20): 4-VO with coinjection of N(ω)-nitro-L-arginine methyl ester (L-NAME, 5 mg/kg, in normal saline, intraperitoneally, 10 min before 4-VO; Sigma, St. Louis, MO, USA), as described (Lin et al., 2010; Zhao et al., 2007), then followed by RIPoC and 48 h and 7 days of reperfusion. (6) The LY + I/R + RIPoC group (n = 20): I/R + RIPoC with injection of LY294002 (LY, 10 μL, 10 mmol/L, in 3% DMSO, intracerebroventricularly, 10 min before 4-VO; Sigma), as
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described in our previous study (Yuan et al., 2011), then followed by RIPoC and 48 h and 7 days of reperfusion. TUNEL assay and Western blot analysis were performed after 48 h of reperfusion. Morris water maze task was performed from the 4th to 7th day after reperfusion. Delayed neuronal death in the CA1 region was examined by Nissl staining after 7 days of reperfusion. 4.3.
Global cerebral ischemia
Global cerebral ischemia was performed as described below using the rat model of 4-VO (Pulsinelli and Brierley, 1979; Pulsinelli et al., 1982) with some modifications (SchmidtKastner et al., 1989; Sun et al., 2006). In brief, the rat was anesthetized with 10% chloral hydrate (350 mg/kg, intraperitoneally). The bilateral vertebral arteries were permanently electrocauterized with a monopolar coagulator through the alar foramens of the first cervical vertebra. The bilateral common carotid arteries (CCAs) were exposed, and ligatures were loosely placed around each artery, without interrupting the carotid blood flow. The bilateral femoral arteries were separated, and the ligature was loosely placed around each artery, without damaging the femoral veins or the femoral nerves. After surgery, the rats were allowed to recover for 24 h. On the second day, CCAs and bilateral femoral arteries were again separated under ether anesthesia by a small animal anesthesia machine. And its head and limbs were attached to a stereotaxic apparatus, which restrained the movements of the conscious rat. The bilateral common carotid and femoral arteries were again separated and exposed. As soon as the rat recovered from the ether anesthesia, one investigator gently retracted the ligatures placed around each artery, and another investigator clamped the bilateral CCAs with atraumatic arterial clips. Global cerebral ischemia was induced by occluding the bilateral CCAs for 8 min. Rats selected for the experiment had the following characteristics: isoelectric level in electroencephalography, no righting reflex, no light reflex, dilated pupils, tonically extended paws within 60 s of 4-VO, and no seizures (Wang et al., 2008; Zhao et al., 2007). The clips were subsequently removed to allow for the reperfusion at the end of ischemia. After the operation, the neck and femoral incisions were closed with 4-0 suture. Rectal temperature was continuously monitored and maintained at 37.0± 0.5 °C until the rat waked up by using a heating pad and lamp during the whole experiment. 4.4.
Remote ischemic postconditioning
RIPoC was performed as described previously in detail (Ren et al., 2009). RIPoC was conducted in the bilateral hind limbs immediately after 8 min of 4-VO by occluding and releasing the bilateral femoral arteries for 3 cycles; each occlusion or release lasted for 15 min. Sham RIPoC consisted of anesthesia and all surgical procedures, except the cycles of bilateral femoral artery occlusion and release. 4.5.
Nissl staining and TUNEL assay
Rats were deeply anesthetized with chloral hydrate and transcardially perfused with 200 mL of normal saline, followed by 300 mL of 4% paraformaldehyde in 0.1 mmol/L phosphate-
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buffered saline at 4 °C (pH 7.4). Brains were quickly removed and fixed in 4% paraformaldehyde at 4 °C for an additional 24 h. After fixation, a brain slice 1–4 mm behind the optic chiasma, which included the bilateral hippocampus, was coronally excised and embedded in paraffin. Embedded brain tissues were sectioned (5 μm thick) for subsequent analyses. Cell counts from the right and the left hippocampus of five sections were averaged to provide mean values. Investigators were blind to the group assignment of the experimental animals during quantification of neuronal characteristics. For TUNEL assay (24 h and 48 h of reperfusion; n = 6), tissue sections were treated according to the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). In brief, sections were incubated for 20 min at room temperature with proteinase K (20 μg/mL in 0.01 mmol/L phosphate-buffered saline). Sections were then incubated in TUNEL reaction mixture, which contained terminal deoxynucleotidyl transferase and fluorescein-labeled dUTP, for 3 h at 37 °C in a humidified chamber in the dark. Sections were then incubated with alkaline phosphatase-conjugated anti-fluorescein in a humidified chamber overnight at 37 °C. Nitroblue tetrazolium and 5bromo-4-chloro-3-indolylphosphate were used to colorize apoptotic cells. For negative controls, sections were incubated without terminal deoxynucleotidyl transferase. The TUNELpositive cells were quantified and expressed as the number of apoptotic cells per 1-mm in the CA1 region. For Nissl staining (7 days of reperfusion; n = 8), tissue sections from each rat were stained with thionin. Neuronal injury was quantified by calculating neuronal density in the CA1 region as described previously (Kirino et al., 1986). The number of neurons per 1-mm linear length was counted in the CA1 region. Normal neurons were characterized as containing Nissl substance in the cytoplasm, loosely organized chromatin, a prominent nucleolus, a full and clear nucleus, an intact outline, and an orderly arrangement. In contrast, damaged neurons were identified by the loss of Nissl substance, cavitation around the nucleus, and the presence of a pyknotic homogenous nucleus (Li et al., 2008; Lu et al., 2010). 4.6.
Morris water maze task
On the 4th day following the induction of ischemia, the Morris water maze task was performed as described previously (Morris, 1984; Yang et al., 2003). The water maze was a circular pool (150 cm in diameter) filled with water (25 ± 1 °C) to a depth of 30 cm. The water's surface was covered with floating black resin beads. An automatic tracking system was used to record escape latency and the swimming path. The water maze, which was conceptually divided into four quadrants, was located in a quiet room that was decorated with visual cues. Rats (n = 8) were placed into the maze at different starting positions, facing the pool wall. A platform (10 cm in diameter) was submerged 2 cm below the surface of the water at the midpoint of one quadrant. During the training trial, rats were placed into the water in one of three randomized starting positions (in a quadrant that did not contain the platform). Each rat was given 120 s to find the invisible platform. If the rat did not succeed within 120 s, it was guided onto the platform with a stick. The rat was then allowed to stay on the platform for 30 s to familiarize itself
with the location of the platform relative to the visual clues. Each rat was given five trials daily for four consecutive days, with an inter-trial interval of 60 min. The escape latency to locate the platform was recorded. On the 7th day following the induction of ischemia, 2 h after the twentieth training trial, the platform was removed from the pool, and the rats were allowed to swim freely for 120 s as a probe trial. The percent of time spent in the target quadrant, which previously contained the platform, and the number of times the rat crossed over the exact location of the former platform, were recorded for each rat. The task assessed how well the rats remembered the location of the hidden platform. Rats were tested in the Morris water maze by investigators who were blinded with respect to the groups. After testing, the rats were sacrificed for Nissl staining. 4.7.
Western blot analysis
After 24 h and 48 h of reperfusion, brains of anesthetized rats (n = 6) were quickly removed at 4 °C. Each brain was placed on ice, and the CA1 region was rapidly isolated and homogenized in RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China). After centrifugation at 12,000 g for 15 min at 4 °C, supernatants were collected. Protein concentrations were determined by the bicinchoninic acid method (Beyotime). An equal amount of protein was loaded in each well and then was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. After blocking with 5% non-fat dry milk, membranes were incubated overnight at 4 °C with the following primary antibody: rabbit polyclonal anti-Bcl-2 (1:500; Millipore, Billerica, CA, USA), rabbit monoclonal antiBax (1:500; Millipore), rabbit polyclonal anti-eNOS (1:1000, Cell Signaling Technology, Beverly, MA, USA), rabbit monoclonal anti-p-eNOS (Ser1177; 1:1000, Cell Signaling Technology), rabbit polyclonal anti-Akt (1:1000, Cell Signaling Technology), rabbit polyclonal anti-p-Akt (Ser473; 1:1000, Cell Signaling Technology), or mouse monoclonal anti-β-actin (1:1000, Beyotime). Membranes were then incubated with secondary antibodies for 1–2 h at room temperature and developed using the BeyoECL Plus kit (Beyotime). Relative band densities were subsequently measured using Quantity One 4.62 (Bio-Rad, Hercules, CA, USA). 4.8.
Statistical analysis
Data are expressed as mean ± standard deviation (SD). Group differences in escape latency in the Morris water maze task were analyzed using a two-way analysis of variance (ANOVA) with repeated measures. The two factors of this analysis were group and training day. Other data were analyzed using a oneway ANOVA with the Student–Newman–Keuls or Dunnett's test. P < 0.05 was considered statistically significant. Data were analyzed using SPSS 16.0 (SPSS, Chicago, IL, USA).
Acknowledgments We are grateful to Prof. Xianghui Zhang for her technical assistance with the Morris water maze task.
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Research Report
Remote ischemic postconditioning protects the brain from global cerebral ischemia/reperfusion injury by up-regulating endothelial nitric oxide synthase through the PI3K/Akt pathway Bei Penga , Qu-lian Guoa,⁎, Zhi-jing Heb , Zhi Yea , Ya-jing Yuana , Na Wanga , Jun Zhouc a
Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, PR China Department of Oral and Maxillofacial Surgery, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, PR China c Center of Experimental Medicine, The Third Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, PR China b
A R T I C LE I N FO
AB S T R A C T
Article history:
Remote ischemic postconditioning (RIPoC) attenuates ischemia/reperfusion (I/R) injury in the
Accepted 16 January 2012
heart, lung and hind limb. RIPoC performed in the hind limb reduces brain injury following
Available online 26 January 2012
focal cerebral ischemia in rats. Whether RIPoC has a neuroprotective effect with respect to global cerebral I/R injury is, however, unknown, and the mechanism of neuroprotection
Keywords:
needs further elucidation. Here we investigated whether RIPoC could reduce global cerebral
Global cerebral ischemia
I/R injury in rats and whether this neuroprotective effect was induced by up-regulating endo-
Remote ischemic postconditioning
thelial nitric oxide synthase (eNOS) through the phosphatidylinositol-3 kinase/Akt (PI3K/Akt)
Delayed neuronal death
pathway. Global cerebral ischemia was performed via 8 min of four-vessel occlusion. Neuronal
Phosphatidylinositol-3 kinase/Akt
density, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-
Endothelial nitric oxide synthase
positive cells and expression of Bcl-2 and Bax in the hippocampal CA1 region were assessed after reperfusion. Morris water maze task was used to quantify spatial learning and memory deficits after reperfusion. The expression of eNOS, phosphorylated eNOS (Ser1177), Akt and phosphorylated Akt (Ser473) in the CA1 region was measured after reperfusion. RIPoC significantly attenuated delayed neuronal death and reduced the spatial learning and memory deficits associated with global cerebral ischemia. Pre-administration of N(ω)-nitro-L-arginine methyl ester (a nonselective NOS inhibitor) significantly abolished the neuroprotective effect of RIPoC. Moreover, pre-administration of LY294002 (a highly selective inhibitor of PI3K) not only significantly reversed the neuroprotective effect of RIPoC, but also obviously inhibited the up-regulation of eNOS induced by RIPoC. Our findings suggest that RIPoC protects the brain against global cerebral I/R injury and that this neuroprotection is mediated by upregulating eNOS through the PI3K/Akt pathway. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: + 86 731 84327413. E-mail address: [email protected] (Q. Guo). Abbreviations: 4-VO, four-vessel occlusion; ANOVA, analysis of variance; CCAs, common carotid arteries; DMSO, dimethyl sulfoxide; eNOS, endothelial nitric oxide synthase; I/R, ischemia and reperfusion; iNOS, inducible nitric oxide synthase; L-NAME, N(ω)-nitro-L-arginine methyl ester; LY, LY294002; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; PI3K, phosphoinositide-3 kinase; RIPoC, remote ischemic postconditioning; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling 0006-8993/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.01.033
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1.
Introduction
Global cerebral ischemia can develop in clinical scenarios that deprive the brain of oxygen and glucose for a short period of time, including carotid endarterectomy, cardiac arrest and cardiopulmonary bypass surgery (Lee et al., 2000; Liang et al., 2008). Rapid reperfusion is the most effective treatment for ischemia, minimizing both structural and functional injuries. Paradoxically, however, restoration of cerebral blood flow causes further damage to the ischemic brain (Frantseva et al., 2001; Tsubota et al., 2010). Many studies have attempted to identify pharmacological or physical interventions that effectively protect the brain against cerebral ischemia and reperfusion (I/R) injury, both global and focal. Ischemic postconditioning, which refers to a short period of sub-lethal ischemia, limits I/R injury in both myocardial and cerebral infarctions when performed immediately or shortly after reperfusion (Chao et al., 2010; Sandu and Schaller, 2010; Wang et al., 2008; Xing et al., 2008). A fascinating new intervention, termed remote ischemic postconditioning (RIPoC), has recently emerged as an effective way to ameliorate I/R injury in the heart, lung and hind limb (Gritsopoulos et al., 2009; Kerendi et al., 2005; Tsubota et al., 2010). Ren et al. (2009) first reported that RIPoC performed in the ipsilateral hind limb can reduce the cerebral infarct size following focal cerebral ischemia. It is, however, not known whether RIPoC can reduce I/R injury following global cerebral ischemia, which has a different pathophysiology than does focal cerebral ischemia. Although several molecular mechanisms are involved in the protective effect of RIPoC against I/R injury in other organs (Andreka et al., 2007; Gritsopoulos et al., 2009; Kerendi et al., 2005), the mechanism of neuroprotection afforded by RIPoC against cerebral I/R injury has not been fully elucidated. Nitric oxide synthase (NOS) plays an important role in physiological and pathological events in the central nervous system. Endothelial NOS (eNOS) is found in a subset of neurons and in the endothelium of cerebral blood vessels. Nitric oxide (NO) generated by eNOS is crucial for vascular function and homeostasis and plays a critical role in the protection of ischemic preconditioning (Chen et al., 2010a, 2010b; Liu et al., 2006; Scorziello et al., 2007) and ischemic postconditioning (Liu et al., 2007) against I/R injury. Using L-arginine or physical activity to up-regulate eNOS increases cerebral blood flow and protects against cerebral ischemia (Lin et al., 2010). The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in signal transduction related to cell growth, proliferation, differentiation, motility, survival and metabolism (Hui et al., 2005). In addition, it is also an important factor in the neuroprotective effect of ischemic postconditioning, as it regulates the downstream anti-apoptotic and survival molecules (Gao et al., 2008). Akt, which is activated by phosphorylation via activated PI3K, phosphorylates eNOS on serine 1177 (p-eNOS), thereby activating this enzyme (Endres et al., 2004; Hashiguchi et al., 2004). Therefore, we speculated that upregulation of eNOS by PI3K/Akt may account for the acquisition of a neuroprotective effect during global cerebral I/R injury following RIPoC. In this study, we investigated whether RIPoC can protect the brain against global cerebral I/R injury in rats and whether
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the neuroprotective effect is associated with up-regulation of eNOS mediated by the PI3K/Akt pathway.
2.
Results
2.1. RIPoC protected against global cerebral ischemia/reperfusion injury RIPoC in the I/R + RIPoC group significantly attenuated global cerebral I/R injury as compared with the I/R group (Figs. 1–2). As shown by Nissl staining (Figs. 1A–B), no significant differences in neuronal density were observed between the sham and RIPoC groups (P > 0.05). In contrast, neuronal density was significantly decreased in the I/R group as compared with the sham group (Fig. 1B). The number of intact neurons in the I/R + RIPoC group was significantly greater than that in the I/R group (Fig. 1B). In the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, TUNEL-positive cells in the CA1 region were analyzed after 24 h and 48 h of reperfusion (Figs. 1C–D). Apoptotic cells were infrequently detected in both the sham and RIPoC groups (Fig. 1D). Compared with the sham group, numerous TUNEL-positive cells were detected in the I/R group (Fig. 1D). RIPoC significantly reversed the increase in TUNEL-positive cells in the I/R + RIPoC group as compared with the I/R group (Fig. 1D). Western blot analysis was used to quantify the expression of apoptosis-related proteins Bcl-2 and Bax in the CA1 region after 24 h and 48 h of reperfusion (Figs. 1E–G). Four-vessel occlusion (4-VO) significantly affected the expression of both Bcl-2 and Bax (Figs. 1F–G). Compared with the I/R group, RIPoC significantly up-regulated the levels of Bcl-2 (Fig. 1F) and significantly down-regulated Bax levels (Fig. 1G) in the I/R + RIPoC group. To investigate whether postconditioning strategies that reduced neuronal death also improved behavioral deficits, we next tested the spatial learning and memory of rats in the Morris water maze task (Fig. 2). During the training phase, significant differences in mean escape latency between both training days [F(3,84) = 200.600, P < 0.01] and between groups [F(3,28) = 27.830, P < 0.01] were detected. In addition, an interaction between the training day and group factors was detected [F(9,84) = 4.280, P < 0.01]. Compared with rats from the sham and RIPoC groups, the I/R group took longer to find the hidden platform on all training days (Fig. 2A). The prolonged escape latency of the I/R group was significantly reduced by RIPoC (Fig. 2A). Concerning spatial learning ability, no significant difference between the sham and RIPoC groups was detected at any stage of the training (P > 0.05; Fig. 2A). During the probing phase, the I/R group made fewer platform crossings than did either the sham or RIPoC group, and RIPoC treatment (in the I/R + RIPoC group) significantly increased the number of crossings over the platform site as compared with the I/R group (Fig. 2B). Differences between the sham and RIPoC groups were not significant (P > 0.05). Finally, the percentage of time spent in the target quadrant was used to estimate retention performance (Fig. 2C). The I/R group spent less time in the target quadrant as compared with either the sham or RIPoC
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Fig. 1 – RIPoC attenuated delayed neuronal death and inhibited neuronal apoptosis in the hippocampal CA1 region induced by global cerebral I/R. (A) Representative Nissl staining of the CA1 neurons in the sham, I/R, I/R+ RIPoC and RIPoC groups after 7 days of reperfusion (400×). (B) Neuronal density of surviving neurons in the CA1 region (n= 8). (C) Representative TUNEL staining of the CA1 neurons in the sham, I/R, I/R + RIPoC and RIPoC groups (400×) after 24 h and 48 h of reperfusion. (D) The number of TUNEL-positive cells in the CA1 region in each group (n = 6). (E) Representative blots of Bcl-2 and Bax of the CA1 region in the sham, I/R and I/R + RIPoC groups after 24 h and 48 h of reperfusion (n = 6). (F, G) Relative protein levels of Bcl-2 and Bax based on band densities of Western blots using β-actin as a control. Data are expressed as mean ± SD. Scale bar: 50 μm. *: P < 0.01 vs. the sham group; #: P < 0.01 vs. the I/R group; @: P < 0.01 vs. the I/R + RIPoC group.
group. A significant difference was observed between the I/R and I/R + RIPoC groups, whereas no significant differences were detected between the I/R + RIPoC and RIPoC groups (P > 0.05, Fig. 2C).
2.2. Inhibition of NOS or PI3K reduced the neuroprotective effect of RIPoC To test whether NOS and the PI3K/Akt pathway are crucial to RIPoC-mediated reduction of delayed neuronal death, we
used pharmacological interventions and pre-treated rats with N(ω)-nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) or LY294002 (LY, a highly selective inhibitor of PI3K) (Fig. 3). Neuronal density was significantly decreased in L-NAME + I/R + RIPoC and LY + I/R + RIPoC groups as compared with the I/R + RIPoC group (Fig. 3B). L-NAME and LY significantly reversed the decrease in the number of TUNEL-positive cells that was observed following RIPoC (Fig. 3D). In the Morris water maze task, pre-administration of L-NAME or LY significantly increased the escape latency
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Fig. 2 – RIPoC reduced spatial learning and memory deficits induced by global cerebral I/R. The Morris water maze was performed to test the spatial learning and memory in the sham, I/R, I/R+ RIPoC and RIPoC groups from 4th to 7th day after reperfusion. (A) Escape latency to find the hidden platform during four consecutive days of training. (B) The number of times the rats crossed over the former platform location during the probe trial. (C) The percent of time spent in the target quadrant during the probe trial. (D) Representative swimming paths during the probe trial for each group. Data are expressed as mean ± SD (n = 8). *: P < 0.01 vs. the sham group; #: P < 0.01 vs. the I/R group.
(Fig. 3E), decreased the number of crossings over the platform site (Fig. 3F) and reduced the percentage of time spent in the target quadrant as compared with values for the I/R + RIPoC group (Fig. 3G).
up-regulated expression of p-eNOS and eNOS after reperfusion (Figs. 4B–C).
3. 2.3. RIPoC induced the up-regulation of eNOS that was mediated by the PI3K/Akt pathway To investigate whether RIPoC induced up-regulation of eNOS in the CA1 region and whether this change was mediated through the PI3K/Akt pathway, we carried out the RIPoC procedure in the presence and absence of L-NAME or LY and then measured the protein levels of p-eNOS, eNOS, p-Akt and Akt in the CA1 region after 48 h of reperfusion (Fig. 4). RIPoC significantly increased the expressions of p-eNOS, eNOS and p-Akt when compared with levels in the sham and I/R groups (Figs. 4B–D). L-NAME significantly reversed the increase in p-eNOS and eNOS that was induced by RIPoC following 4-VO as compared with amounts in the I/R + RIPoC group (Figs. 4B–C). Moreover, LY not only significantly prevented the activation of p-Akt that was induced by RIPoC following 4-VO (Fig. 4D), but also significantly reversed the
Discussion
In the present study, 3 cycles of 15-min occlusion/15-min release of the bilateral femoral arteries immediately after 4-VO was selected as the RIPoC protocol. This approach was based on the previous research of Ren et al. (2009), who first expanded the concept of conventional ischemic postconditioning to include remote ischemic postconditioning, which was induced in a distant non-vital organ, in a rat model of focal cerebral I/R. The present study described and investigated the neuroprotective role of RIPoC after global cerebral ischemia via 4-VO. These results demonstrated that RIPoC attenuated delayed neuronal death and spatial learning and memory deficits induced by global cerebral I/R. This neuroprotective effect is apparently involved in regulating the apoptotic proteins Bcl-2 and Bax. Furthermore, inhibition of NOS with L-NAME abolished the attenuation of global cerebral I/R injury provided by RIPoC. Pre-treatment with LY not only prevented the neuroprotective
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Fig. 4 – RIPoC induced the up-regulation of eNOS in the hippocampal CA1 region and this up-regulation was mediated by the PI3K/Akt pathway. Representative Western blot (A) and quantitative analysis of p-eNOS (B), eNOS (C), p-Akt (D) and Akt (E) expressions of the CA1 region of rats in the sham, I/R, I/R + RIPoC, L-NAME + I/R + RIPoC, and LY + I/R + RIPoC groups after 48 h of reperfusion. L-NAME reversed the RIPoC-induced increase in p-eNOS and eNOS following 4-VO as compared with the I/R + RIPoC group. Moreover, LY not only significantly prevented the activation of p-Akt that was induced by RIPoC following 4-VO, but also significantly reversed the up-regulated expression of p-eNOS and eNOS after reperfusion. Data are expressed as mean ± SD (n = 6). *: P < 0.01 vs. the sham group; #: P < 0.01 vs. the I/R group; @: P < 0.01 vs. the I/R + RIPoC group.
effect of RIPoC, but also inhibited RIPoC-induced up-regulation of eNOS in the CA1 region after reperfusion. The brain is intrinsically vulnerable to global ischemia, especially in the CA1 region of the hippocampus (Connell et al., 2011; Lee et al., 2000). In our experiments, RIPoC effectively inhibited neuronal cell apoptosis after 24 and 48 h of reperfusion and reduced delayed neuronal death after 7 days of reperfusion in the CA1 region. Global cerebral ischemia, which leads to the most extensive neuronal damage in the CA1 region, results in a deficit in spatial learning and memory (Wang et al., 2008). Thus, the Morris water maze task was used to quantify spatial learning and memory deficits of rats subjected to global cerebral ischemia. Four-VO significantly impaired spatial learning and memory. RIPoC decreased the
escape latency, increased the number of platform site crossings, and increased the time spent in the target quadrant. It seems, therefore, that neurons rescued by the RIPoC treatment maintained at least some of their functional parameters. Our results are consistent with those of Ren et al. (2009), which showed that RIPoC is protective in the rat model of focal cerebral ischemia (middle cerebral artery occlusion), an occurrence that has a pathophysiology that is very different from that of global cerebral ischemia (Hashiguchi et al., 2004). NO and its derivatives can have multiple effects that affect neuronal death in different ways. It should be mentioned that the roles of NO and NOS during I/R are controversial (Brown, 2010). NO from NOS has both neurotoxic and neuroprotective roles during cerebral I/R (Endres et al., 2004; Scorziello et al.,
Fig. 3 – Inhibition of NOS or PI3K reversed the neuroprotective effects of RIPoC. (A) Representative Nissl staining of the hippocampal CA1 neurons in the I/R, I/R + RIPoC, L-NAME + I/R + RIPoC and LY + I/R + RIPoC groups after 7 days of reperfusion (400×). (B) Neuronal density of surviving neurons in the CA1 region (n = 8). (C) Representative TUNEL staining of the CA1 neurons in the I/R, I/R + RIPoC, L-NAME+ I/R +RIPoC and LY + I/R + RIPoC groups (400×) after 48 h of reperfusion. (D) The number of TUNEL-positive cells in the CA1 region (n= 6). (E, F, G, H) The spatial learning and memory were tested in the I/R, I/R + RIPoC, L-NAME + I/R + RIPoC and LY + I/R+ RIPoC groups from 4th to 7th day after reperfusion (n= 8). (E) Escape latency to find the hidden platform during four consecutive days of training. (F) The number of times the rats crossed over the former platform location during the probe trial. (G) The percent of time spent in the target quadrant during the probe trial. (H) Representative swimming paths during the probe trial for each group. Data are expressed as mean± SD. Scale bar: 50 μm. #: P < 0.01 vs. the I/R group; @: P < 0.01 vs. the I/R + RIPoC group; $: P < 0.05 vs. the I/R + RIPoC group.
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2007). Excessive production of NO during the early minutes of reperfusion can produce I/R-induced injury via lipid peroxidation, DNA damage and pro-apoptotic effects (Liu et al., 2007; Zhao et al., 2010). In contrast, NO is protective based on its ability to cause vasodilatation, inhibit platelet plug formation and reduce the inflammatory response, as well as its anti-apoptotic actions (Chen et al., 2010a, 2010b; Zhao et al., 2007). NO can induce protection via cyclic guanosine monophosphate-mediated vasodilation and mitochondrial permeability transition inhibition by reactive oxygen and nitrogen species-mediated activation of the NF-κB and mitogen-activated protein kinase pathways or via S-nitros(yl)ation of caspases or the N-methyl-D-aspartate receptor (Brown, 2010; Hashiguchi et al., 2004). The cellular effects of NO may be dependent on its concentration, site of release and duration of action (Liu et al., 2009; Rastaldo et al., 2007). We showed that L-NAME, a nonselective NOS inhibitor, abolished the neuroprotective effect induced by RIPoC following 4-VO. This strongly suggests that, in the case of the neuroprotective effect of RIPoC against global cerebral I/R injury, the neuroprotective role of NO and NOS is more relevant than their neurotoxic role. The three isoforms of NOS play different roles in ischemic brain damage: eNOS protects (probably by maintaining vasodilation and promoting angiogenesis), neuronal NOS (nNOS) mediates neuronal damage (probably by leading to peroxynitrite production) and inducible NOS (iNOS) can play protective or damaging roles or both (Hashiguchi et al., 2004). nNOS knockout mice were partially protected against ischemia-induced neuronal death (Brown, 2010). Three different strains of iNOS knockout showed no effect of iNOS on infarct size after transient focal brain ischemia, and there was no induction of iNOS expression by ischemia (Pruss et al., 2008). NO induced by eNOS can also protect neurons against focal ischemia in the brain via the vasodilatory action of NO (Hashiguchi et al., 2004). Therefore, we focused on the expression of eNOS after reperfusion instead of iNOS and nNOS in the present study. NO produced by eNOS is important in vascular signaling and tone regulation and leukocyte–endothelial interactions (Lin et al., 2010). eNOS-deficient mice are hypertensive and have an increased infarct volume after a stroke (Endres et al., 2004; Sawada and Liao, 2009). In contrast, transgenic mice in which eNOS is overexpressed are resistant to stress-induced hypotension, inflammation and stroke (Yamashita et al., 2000). Up-regulation of eNOS in neurons and endothelium was reported to be protective, and the mechanism that leads to this effect may involve anti-apoptosis (De Palma et al., 2008). In the current study, RIPoC significantly up-regulated the expression of eNOS and p-eNOS in the CA1 region after global cerebral I/R, and inhibition of NOS significantly reversed the neuroprotective effect of RIPoC on global cerebral I/R injury. These results suggest that eNOS and NO are involved in the neuroprotective effect of RIPoC in the rat model of global cerebral ischemia. L-NAME is an arginine analog commonly used as a potent and reversible L-arginine competitive inhibitor of NOS. Nevertheless, it was demonstrated that L-NAME reduced the up-regulation of eNOS and p-eNOS expressions induced by RIPoC in the CA1 region. And previous studies showed that pretreatment with L-NAME inhibited the limb ischemic preconditioning induced increase of eNOS activities in the hippocampus and serum in global cerebral I/R injury (Zhao et al., 2007), reduced the upregulated expressions of eNOS and p-eNOS by eNOS gene
transfer in myocardial infarction (Chen et al., 2007, 2010a, 2010b), decreased the increased level of eNOS in liver by ischemic postconditioning in liver warm I/R injury (Guo et al., 2011) and abolished the induction of the expression of eNOS by Ginsenoside Rb1 in myocardial I/R injury (Xia et al., 2011). However, the mechanism remains unclearly understood. We speculated that L-NAME intraperitoneally may reduce the expression and activation of eNOS in the CA1 region by some other signaling pathways during the reperfusion of the brain in this study. Therefore, future studies need to address why L-NAME reduced RIPoC-induced eNOS expression and phosphorylation in the CA1 region. Among the several signaling pathways that up-regulate eNOS following various pharmacological stimuli, we were particularly interested in the PI3K/Akt pathway. The PI3K/Akt pathway is involved in the neuroprotective effect of ischemic postconditioning (Yuan et al., 2011; Zhou et al., 2011). In the rat model of global cerebral ischemia in this study, RIPoC increased Akt phosphorylation, and the PI3K inhibitor LY, which prevents the phosphorylation and activation of Akt, significantly inhibited RIPoC-induced neuroprotection. Akt phosphorylates eNOS at Ser1177 in neurons and endothelial cells, thereby activating the enzyme (Hashiguchi et al., 2004; Lin et al., 2010). The activation of eNOS by Akt might contribute to the important physiological effects on apoptosis, cell attachment and cell proliferation (Dimmeler et al., 1999). In our study, inhibition of the PI3K/Akt pathway significantly prevented the RIPoC-induced increase of eNOS and p-eNOS expressions in the CA1 region after reperfusion. In addition, the PI3K/Akt pathway-mediated up-regulation of eNOS is required for the anti-apoptotic effect of insulin, estrogen and statins in stroke (Endres et al., 2004). PI3K/Akt signaling leading to eNOS activation in neurons and endothelial cells contributes to ischemic tolerance in the rat hippocampus (Lin et al., 2010). The data in this study suggest that the activation of the PI3K/Akt pathway by protective stimuli in the hind limbs not only was involved in the neuroprotective effect of RIPoC, but also mediated the up-regulation of eNOS in the CA1 region that was induced by RIPoC. In the present study, we assessed whether RIPoC treatment alone has an effect on delayed neuronal death and spatial learning and memory. Our data show that RIPoC had an insignificant effect on neuronal density, the TUNEL assay results and the Morris water maze task if 4-VO had not been previously carried out. We therefore have not assessed the effect of RIPoC treatment alone on the expression of apoptosisrelated proteins Bcl-2 and Bax in the CA1 region. Furthermore, although vehicle-only treatments were not included in the investigation of the potential role and relationship of the PI3K/Akt pathway and eNOS in the neuroprotective effect of RIPoC, a single injection of normal saline or dimethyl sulfoxide (DMSO) has been reported to have no effect on the NOS or the PI3K/Akt pathway (Yuan et al., 2011; Zhou et al., 2011). In fact, DMSO does not affect PI3K and is commonly used as a solvent for pharmacological agents (Liu et al., 2010). Therefore, we believe that L-NAME and LY, and not their associated solvents, reversed the RIPoC-mediated neuroprotective effects. RIPoC is a simple and harmless method that provides a new tool to prevent I/R injury. Shown clearly were the feasibility and safety in application of RIPoC as an adjunct to current
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reperfusion strategies. It would be practical to create limb ischemia by placing a tourniquet on an extremity for a brief period of time and then releasing it after cerebral reperfusion (Li et al., 2006; Ren et al., 2009). In conclusion, repetitive brief sub-lethal I/R in the hind limbs may result in the activation of molecular pathways responsible for RIPoC, thereby protecting the brain from global cerebral I/R injury. RIPoC appeared to attenuate delayed neuronal death and ameliorate spatial learning and memory deficits that resulted from global cerebral ischemia. This neuroprotective effect was mediated by up-regulating eNOS through the PI3K/Akt pathway.
4.
Experimental procedures
4.1.
Animals
Adult male Sprague–Dawley rats (200–250 g), obtained from the Experimental Animal Centre of Central South University, were used for this study according to the Guide for the Care and Use of Laboratory Animals (Zimmermann, 1983). The animal experiments were approved by the animal ethics committee of Xiangya Hospital Central South University. All the rats were housed in cages in a temperature-controlled (24 ± 1 °C) room and were maintained under diurnal lighting conditions (12 h light/dark). They had free access to food and water. Pain and suffering to the rats were minimized. Prior to the experiments, rats were fed in the laboratory animal room for 2 days to allow for adaptation to the environment. 4.2.
Experimental protocols
To investigate the neuroprotective effects of RIPoC against global cerebral I/R injury, rats were randomly divided to four groups: (1) the sham group (n = 20): rats were subjected to sham operations without 4-VO and RIPoC. (2) The I/R group (n = 32): rats were subjected to 4-VO followed by 24 h, 48 h and 7 days of reperfusion and sham RIPoC. (3) The I/R + RIPoC group (n = 32): rats were subjected to 4-VO followed by RIPoC and 24 h, 48 h and 7 days of reperfusion. (4) The RIPoC group (n = 32): rats were subjected to sham 4-VO followed by RIPoC and 24 h, 48 h and 7 days of reperfusion. TUNEL assay and Western blot analysis were performed after 24 h and 48 h of reperfusion. The spatial learning and memory were tested in Morris water maze task from the 4th to 7th day after reperfusion. Delayed neuronal death in the hippocampal CA1 region was examined by Nissl staining after 7 days of reperfusion. To investigate the potential roles and relationship of PI3K/Akt pathway and eNOS in neuroprotection of RIPoC, rats were randomly divided to additional two groups: (5) the L-NAME + I/R + RIPoC group (n = 20): 4-VO with coinjection of N(ω)-nitro-L-arginine methyl ester (L-NAME, 5 mg/kg, in normal saline, intraperitoneally, 10 min before 4-VO; Sigma, St. Louis, MO, USA), as described (Lin et al., 2010; Zhao et al., 2007), then followed by RIPoC and 48 h and 7 days of reperfusion. (6) The LY + I/R + RIPoC group (n = 20): I/R + RIPoC with injection of LY294002 (LY, 10 μL, 10 mmol/L, in 3% DMSO, intracerebroventricularly, 10 min before 4-VO; Sigma), as
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described in our previous study (Yuan et al., 2011), then followed by RIPoC and 48 h and 7 days of reperfusion. TUNEL assay and Western blot analysis were performed after 48 h of reperfusion. Morris water maze task was performed from the 4th to 7th day after reperfusion. Delayed neuronal death in the CA1 region was examined by Nissl staining after 7 days of reperfusion. 4.3.
Global cerebral ischemia
Global cerebral ischemia was performed as described below using the rat model of 4-VO (Pulsinelli and Brierley, 1979; Pulsinelli et al., 1982) with some modifications (SchmidtKastner et al., 1989; Sun et al., 2006). In brief, the rat was anesthetized with 10% chloral hydrate (350 mg/kg, intraperitoneally). The bilateral vertebral arteries were permanently electrocauterized with a monopolar coagulator through the alar foramens of the first cervical vertebra. The bilateral common carotid arteries (CCAs) were exposed, and ligatures were loosely placed around each artery, without interrupting the carotid blood flow. The bilateral femoral arteries were separated, and the ligature was loosely placed around each artery, without damaging the femoral veins or the femoral nerves. After surgery, the rats were allowed to recover for 24 h. On the second day, CCAs and bilateral femoral arteries were again separated under ether anesthesia by a small animal anesthesia machine. And its head and limbs were attached to a stereotaxic apparatus, which restrained the movements of the conscious rat. The bilateral common carotid and femoral arteries were again separated and exposed. As soon as the rat recovered from the ether anesthesia, one investigator gently retracted the ligatures placed around each artery, and another investigator clamped the bilateral CCAs with atraumatic arterial clips. Global cerebral ischemia was induced by occluding the bilateral CCAs for 8 min. Rats selected for the experiment had the following characteristics: isoelectric level in electroencephalography, no righting reflex, no light reflex, dilated pupils, tonically extended paws within 60 s of 4-VO, and no seizures (Wang et al., 2008; Zhao et al., 2007). The clips were subsequently removed to allow for the reperfusion at the end of ischemia. After the operation, the neck and femoral incisions were closed with 4-0 suture. Rectal temperature was continuously monitored and maintained at 37.0± 0.5 °C until the rat waked up by using a heating pad and lamp during the whole experiment. 4.4.
Remote ischemic postconditioning
RIPoC was performed as described previously in detail (Ren et al., 2009). RIPoC was conducted in the bilateral hind limbs immediately after 8 min of 4-VO by occluding and releasing the bilateral femoral arteries for 3 cycles; each occlusion or release lasted for 15 min. Sham RIPoC consisted of anesthesia and all surgical procedures, except the cycles of bilateral femoral artery occlusion and release. 4.5.
Nissl staining and TUNEL assay
Rats were deeply anesthetized with chloral hydrate and transcardially perfused with 200 mL of normal saline, followed by 300 mL of 4% paraformaldehyde in 0.1 mmol/L phosphate-
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buffered saline at 4 °C (pH 7.4). Brains were quickly removed and fixed in 4% paraformaldehyde at 4 °C for an additional 24 h. After fixation, a brain slice 1–4 mm behind the optic chiasma, which included the bilateral hippocampus, was coronally excised and embedded in paraffin. Embedded brain tissues were sectioned (5 μm thick) for subsequent analyses. Cell counts from the right and the left hippocampus of five sections were averaged to provide mean values. Investigators were blind to the group assignment of the experimental animals during quantification of neuronal characteristics. For TUNEL assay (24 h and 48 h of reperfusion; n = 6), tissue sections were treated according to the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). In brief, sections were incubated for 20 min at room temperature with proteinase K (20 μg/mL in 0.01 mmol/L phosphate-buffered saline). Sections were then incubated in TUNEL reaction mixture, which contained terminal deoxynucleotidyl transferase and fluorescein-labeled dUTP, for 3 h at 37 °C in a humidified chamber in the dark. Sections were then incubated with alkaline phosphatase-conjugated anti-fluorescein in a humidified chamber overnight at 37 °C. Nitroblue tetrazolium and 5bromo-4-chloro-3-indolylphosphate were used to colorize apoptotic cells. For negative controls, sections were incubated without terminal deoxynucleotidyl transferase. The TUNELpositive cells were quantified and expressed as the number of apoptotic cells per 1-mm in the CA1 region. For Nissl staining (7 days of reperfusion; n = 8), tissue sections from each rat were stained with thionin. Neuronal injury was quantified by calculating neuronal density in the CA1 region as described previously (Kirino et al., 1986). The number of neurons per 1-mm linear length was counted in the CA1 region. Normal neurons were characterized as containing Nissl substance in the cytoplasm, loosely organized chromatin, a prominent nucleolus, a full and clear nucleus, an intact outline, and an orderly arrangement. In contrast, damaged neurons were identified by the loss of Nissl substance, cavitation around the nucleus, and the presence of a pyknotic homogenous nucleus (Li et al., 2008; Lu et al., 2010). 4.6.
Morris water maze task
On the 4th day following the induction of ischemia, the Morris water maze task was performed as described previously (Morris, 1984; Yang et al., 2003). The water maze was a circular pool (150 cm in diameter) filled with water (25 ± 1 °C) to a depth of 30 cm. The water's surface was covered with floating black resin beads. An automatic tracking system was used to record escape latency and the swimming path. The water maze, which was conceptually divided into four quadrants, was located in a quiet room that was decorated with visual cues. Rats (n = 8) were placed into the maze at different starting positions, facing the pool wall. A platform (10 cm in diameter) was submerged 2 cm below the surface of the water at the midpoint of one quadrant. During the training trial, rats were placed into the water in one of three randomized starting positions (in a quadrant that did not contain the platform). Each rat was given 120 s to find the invisible platform. If the rat did not succeed within 120 s, it was guided onto the platform with a stick. The rat was then allowed to stay on the platform for 30 s to familiarize itself
with the location of the platform relative to the visual clues. Each rat was given five trials daily for four consecutive days, with an inter-trial interval of 60 min. The escape latency to locate the platform was recorded. On the 7th day following the induction of ischemia, 2 h after the twentieth training trial, the platform was removed from the pool, and the rats were allowed to swim freely for 120 s as a probe trial. The percent of time spent in the target quadrant, which previously contained the platform, and the number of times the rat crossed over the exact location of the former platform, were recorded for each rat. The task assessed how well the rats remembered the location of the hidden platform. Rats were tested in the Morris water maze by investigators who were blinded with respect to the groups. After testing, the rats were sacrificed for Nissl staining. 4.7.
Western blot analysis
After 24 h and 48 h of reperfusion, brains of anesthetized rats (n = 6) were quickly removed at 4 °C. Each brain was placed on ice, and the CA1 region was rapidly isolated and homogenized in RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China). After centrifugation at 12,000 g for 15 min at 4 °C, supernatants were collected. Protein concentrations were determined by the bicinchoninic acid method (Beyotime). An equal amount of protein was loaded in each well and then was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. After blocking with 5% non-fat dry milk, membranes were incubated overnight at 4 °C with the following primary antibody: rabbit polyclonal anti-Bcl-2 (1:500; Millipore, Billerica, CA, USA), rabbit monoclonal antiBax (1:500; Millipore), rabbit polyclonal anti-eNOS (1:1000, Cell Signaling Technology, Beverly, MA, USA), rabbit monoclonal anti-p-eNOS (Ser1177; 1:1000, Cell Signaling Technology), rabbit polyclonal anti-Akt (1:1000, Cell Signaling Technology), rabbit polyclonal anti-p-Akt (Ser473; 1:1000, Cell Signaling Technology), or mouse monoclonal anti-β-actin (1:1000, Beyotime). Membranes were then incubated with secondary antibodies for 1–2 h at room temperature and developed using the BeyoECL Plus kit (Beyotime). Relative band densities were subsequently measured using Quantity One 4.62 (Bio-Rad, Hercules, CA, USA). 4.8.
Statistical analysis
Data are expressed as mean ± standard deviation (SD). Group differences in escape latency in the Morris water maze task were analyzed using a two-way analysis of variance (ANOVA) with repeated measures. The two factors of this analysis were group and training day. Other data were analyzed using a oneway ANOVA with the Student–Newman–Keuls or Dunnett's test. P < 0.05 was considered statistically significant. Data were analyzed using SPSS 16.0 (SPSS, Chicago, IL, USA).
Acknowledgments We are grateful to Prof. Xianghui Zhang for her technical assistance with the Morris water maze task.
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