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Keywords:

  • forkhead transcription factors;
  • hippocampus;
  • hypoxic pre-conditioning;
  • ischemia;
  • ischemic tolerance;
  • protein kinase B

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

J. Neurochem. (2010) 114, 897–908.

Abstract

It is well established that pre-conditioning protects neuronal injury against ischemia. However, the molecular mechanisms underlying ischemic tolerance are not completely understood. The purpose of the present study was to investigate the role of Akt/forkhead transcription factor, class O (FoxO) pathway in hypoxic pre-conditioning (HPC) using a newly developed HPC to transient global cerebral ischemia (tGCI) model in adult rats. HPC for 30–120 min significantly reduced cell death in the CA1 subregion after 10 min of tGCI. HPC was effective only when applied 1–4 days before ischemia. The maximum protection was observed with 30 min of hypoxia and 1 day interval between hypoxia and ischemia. The phosphorylated Akt and FoxOs measured by western blot and immunohistochemistry were significantly increased after hypoxia-ischemia except for a transient decrease in the HPC group. Lateral ventricular infusion of LY294002 before HPC blocked the increase in phosphorylated Akt and FoxOs and increased neuronal damage in HPC animals. These results suggest that pre-exposure to hypoxia induces protection against tGCI in adult rats. Activation of Akt results in the inactivation of FoxOs which may mediate ischemic tolerance after HPC.

Abbreviations used:
FoxO

forkhead transcription factor, class O

GFAP

glial fibrillary acidic protein

HPC

hypoxic pre-conditioning

IT

ischemic tolerance

MAP-2

microtubule associated protein-2

MCAO

middle cerebral artery occlusion

NeuN

neuronal nuclei

PBS

phosphate buffer saline

PI3K

phosphoinositide 3-kinase

tGCI

transient global cerebral ischemia

Accumulating evidence indicated that sublethal pre-treatments can provide neuroprotective adaptation against subsequent severe ischemia in the brain (Kitagawa et al. 1990; Kapinya 2005; Ran et al. 2005). This process has been termed pre-conditioning or ischemic tolerance (IT). Evidence for the existence of ischemic pre-conditioning in humans has been reported (Moncayo et al. 2000; Schaller 2005). The major goal of studying IT is to identify the underlying endogenous protective signaling cascades, with the long-term goal to allow therapeutic augmentation of the endogenous protective mechanisms in cerebral ischemia and possibly to induce a protected state of the brain in conditions in which brain ischemia can be anticipated, for example, during surgery of the heart or cardiac arrest upon resuscitation.

To study the mechanisms of IT, clinically relevant models are needed. Experimentally, combinations of various types of ischemic insults have been developed to study IT (Kapinya 2005). Although ischemic pre-conditioning offers protection against ischemia in animal models, it could not be applied to patients for ethics reasons. On the other hand, moderate hypoxia, which does not cause neuronal death and may be safer to be applied in clinical practice, becomes an attractive method in animal research for IT. However, studies on hypoxia-induced IT mainly focused on focal ischemic models in the neonatal rats (Gidday et al. 1994; Ota et al. 1998) or adult mice (Bernaudin et al. 2002), it is not known whether hypoxia protects the adult rat brain against transient global cerebral ischemia (tGCI).

It has been suggested that hypoxic pre-conditioning (HPC) in the neonatal rat brain enhances intracellular pro-survival signaling pathways via altering gene expression and/or post-translational modification of signaling molecules (Ran et al. 2005). Akt (protein kinase B) is a protein kinase involved in survival signals as a downstream kinase of phosphoinositide 3-kinase (PI3K) in growth factor-mediated signaling cascades. Akt pathway is one of the pathways activated by ischemic pre-conditioning in the adult rodent brain, likely playing a critical role in promoting neuronal survival after ischemia (Yano et al. 2001; Yin et al. 2005). However, whether Akt pathway plays a role in hypoxia-induced tolerance to tGCI injury in the adult rat brain is not clear. Previous studies have indicated that activation of Akt promotes cell survival through phosphorylation of a series of substrates, such as class O members of the forkhead transcription factor family (FoxOs) (Brunet et al. 1999). FoxO is a mammalian homologue of DAF-16, which is known to regulate the life span of Caenorhabditis elegans and includes subfamilies of forkhead transcription regulators, including FKHR (FoxO1), FKHRL1 (FoxO3a), and AFX (FoxO4) (Lin et al. 1997). It has been documented that the activation of FoxOs is involved in the mechanisms of cell death induced by ischemia (Kawano et al. 2002; Shioda et al. 2007b). However, whether the inactivation of FoxOs is involved in the mechanisms of IT brought about by HPC remains unknown.

The present study aims to develop an animal model of hypoxia-induced tolerance to tGCI in adult rats and further investigate whether Akt is activated and its substrates, FoxOs, are inactivated in this kind of pre-conditioning. We present for the first time functional evidence that phosphorylation of Akt and FoxOs are important mediators of protection in HPC.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animal model

Experiments were performed on adult male Wistar rats weighing 250–300 g (Southern Medical University, Guangdong, China). Animals were treated according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animal (Publication No. 80–23) revised 1996. Guangzhou Medical College Committee on Use and Care of Animals closely monitored compliance with the NIH regulations.

Hypoxia was induced by placing the rats in a sealed plastic chamber of 9000 cm3 volume through which air containing 8% O2 and 92% N2 flows continuously at a temperature of 23–25°C. A total gas flow was 200 mL/min and no more than three animals were placed in the chamber at any given time.

Transient global ischemia was induced using the four-vessel occlusion method (Pulsinelli and Brierley 1979). Briefly, the animals were anesthetized with chloral hydrate (350 mg/kg, i.p.). Vertebral arteries were electrocauterized, and common carotid arteries were isolated. A teflon/silastic occluding device was placed loosely around each carotid artery without interrupting carotid blood flow. Forebrain ischemia was induced in the rats which are awake, 24 h after the surgery by occluding both common carotid arteries for 10 min. After occlusion, rats that lost their righting reflex within 1 min and pupils dilated were selected for experiments. Rectal temperature was maintained at 37–38°C throughout the procedure. Sham-operated rats were performed using the same surgical procedures except that the arteries were not occluded.

To assess potential neuronal damage after hypoxic exposure, six rats per group underwent hypoxia as described above without subsequent ischemia. To evaluate the effects of different hypoxic duration, animals received 30 min, 60 min, 120 min, or 180 min hypoxia 2 days before ischemia (the number of rats in each group was 9, 8, 6, and 7, respectively). To determine the best hypoxia-ischemic interval, a 30 min hypoxia was performed 1–5 days before ischemia (the number of rats in each group was 9, 9, 7, 6, and 7, respectively).

Administration of LY294002

LY294002 (Cell Signaling Technology, Beverly, MA, USA. 50 mmol/L in 25% dimethylsulfoxide sulfoxide in 0.01 M phosphate buffer saline (PBS, pH 7.4)) (Endo et al. 2006) or the vehicle (25% dimethylsulfoxide in PBS) was injected intracerebroventricularly 30 min before HPC or ischemia (10 μL, i.c.v., bregma: 1.5 mm lateral, 0.8 mm posterior, 4.0 mm deep). To evaluate the toxicity of LY294002 to the cells of hippocampus, six animals without hypoxia or ischemia were treated with 10 μL LY294002 intracerebroventricularly.

Histology

Animals were perfused intracardially with normal saline, followed by 4% paraformaldehyde in PBS under anesthesia. Brains were removed and post-fixed for 24 h at 4ºC, and immersed in 15%, 30% sucrose in the same fixative for cytoprotection. Coronal free-floating sections were cut at 30 μm using a cryotome (Thermo, Runcorn, Cheshire, UK). Sections selected from the dorsal hippocampus (between AP 4.8–5.8 mm, interaural or AP −3.3 to 3.4 mm, Bregma) were stained with Cresyl violet and examined under a light microscope (×660). The surviving cells numbers in the CA1 layer 1-mm length were counted. All of the data for cell counting were collected from two specific regions in the CA1 layer. Cell damage was quantified bilaterally in sections from each brain and assessed in double blind. Four sections for each animal were evaluated.

Western blotting

The rats were killed at 0, 4, 24 and 48 h (n = 4 in each group) after reperfusion or at 24 h after hypoxia. The CA1 subregion protein extraction was performed as described previously (Yano et al. 2001). Western blotting analyses were performed as described previously (Endo et al. 2006). Primary antibodies included phospho-Akt (Ser473) (1 : 1000), Akt(1 : 8000), phospho-FKHR (Ser256) (1 : 1000), FKHR (1 : 1000), phospho-FKHRL1 (Ser253) (1 : 4000), FKHRL1 (1 : 1000), phospho-AFX (Ser197) (1 : 4000), AFX (1 : 1000), and ß-actin (1 : 1000). All antibodies were prepared from rabbits except the antibody of phospho-Akt which was from mouse. All antibodies were purchased from Cell Signaling Technology, except for phospho-FKHR, phospho-FKHRL1 and phospho-AFX which were purchased from Signalway Antibody (Pearland, TX, USA).

Immunohistochemistry

The rats were killed at 0, 4, 24, 48 h, and 7 days (n = 6 in each group) after reperfusion or at 24 h after hypoxia. Single-lable immunohistochemistry was performed as described previously (Wang et al. 2010). The primary antibodies used in these studies include phospho-Akt (1 : 200; Cell Signaling), phospho-FKHR (1 : 400; Signalway Antibody), phospho-FKHRL1 (1 : 400; Signalway Antibody) and phospho-AFX (1 : 400; Signalway Antibody).

Double-fluorescent immunohistochemistry was conducted to demonstrate the cell types and the exact position that expressed phospho-Akt and phospho-FKHR. Hoechst 33528 was used to confirm the nuclear expression of the proteins. Neuronal nuclei (NeuN), microtubule associated protein-2 (MAP-2) and glial fibrillary acidic protein (GFAP) was used to identify neuronal nuclei, neuronal cell bodies and dendrites, and astrocytes, respectively. Double-fluorescent immunohistochemistry was performed as described previously (Yuan et al. 2009; Wang et al. 2010). Antibodies used in these studies include phospho-Akt (1 : 50; Cell Signaling), phospho-FKHR (1 : 100; Signalway Antibody), NeuN (1 : 400; Chemicon, Tenecula, CA, USA), MAP-2 (1 : 100; Chemicon), GFAP (1 : 100; Chemicon), Cy3-conjugated goat anti-mouse IgG antibody (1 : 50; Invitrogen, Carlsbad, CA, USA) and FITC-conjugated goat anti-rabbit antibody (1 : 100; Invitrogen). Slides analyzed with a confocal laser microscope (Leica Microsystems, Wetzlar, Hessen, Germany).

Data analyses

Densitometric analysis for the quantification of the bands or the staining strength was performed using image analysis software (WCIF ImageJ, and Quantity One, Bio-Rad Laboratories, Inc. Hercules, CA, USA, respectively). Immunopositive cells were quantified in four sections of each animal under high power microscope (×1200). For each section, the immunoreactive cells number was estimated by counting the cells in which the reaction product was present within a clear and regular-shaped cytoplasmic border from five random fields in the CA1 subregion. Statistical analyses were performed with the Statistical Package for Social Sciences for Windows, version 11.5 (SPSS, Inc, Chicago, Illinois, USA). Measurement data were summarized by mean ± SD, and the statistical significance was determined by one-way anova or the two-tailed Student’s t-test. < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Effects of hypoxic preconditioning on cell damage

Sham-operated animals had no morphological abnormality throughout the observation period (n = 6, Fig. 1a, i and ii). Up to 120 min of hypoxia, there were no any detectable damage at the light microscopic level in pyramidal cells of the hippocampus when it was examined 7 days after hypoxia without ischemia (n = 6, Fig. 1a, iii and iv). After 180 min of hypoxia without ischemia, only 50% of the animals survived during hypoxia, and cell damage to the CA1 sector became detectable (n = 6, Fig. 1a, v and vi), including cell body swelling, and Nissl substance decrease, but there were no evident cell loss (Fig. 1a, vi). Cell damage was quite evident in the CA1 pyramidal cells on day 7 after 10 min of tGCI (n = 8, Fig. 1a, vii and viii), including cell body severe shrinkage and darkening, Nissl substance loss, and nuclear condensation or disaggregation (Fig. 1a, viii). Compared with sham-operated group, 10 min of tGCI destroyed 79.69% of CA1 neurons.

image

Figure 1.  Effects of hypoxic pre-conditioning on neuronal damage in the CA1 subregion after transient global cerebral ischemia (tGCI). (a) Photomicrograph of sections showing Cresyl violet staining of the hippocampus. The pictures on the right are magnified from the square areas on the left. Sham group (i, ii; n = 6), 120-min hypoxia only group (iii, iv; n = 6), 180-min hypoxia only group (v, vi; n = 6), tGCI control group, histology was performed 7 days after reperfusion in rats subjected to10 min of tGCI (vii, viii; n = 8), hypoxic preconditioning (HPC) + Is group, histology was performed 7 days after reperfusion in rats subjected to10 min of tGCI with 30 min hypoxia 1-day interval (ix, x; n = 9). Sham, sham-operated rats. Scale bar: i,iii,v,vii,ix: 250 μm, ii,iv,vi,viii,x: 25 μm. (b) Numbers of surviving cell in the CA1 subregion after different durations of hypoxia. Animals were pretreated with hypoxia for 30 min, 60 min, 120 min, or 180 min, 2 days later, tGCI was induced for 10 min (the number of rats in each group was 9, 8, 6, and 7, respectively). (c) Numbers of surviving cell in the CA1 subregion after different pre-treatment intervals. Rats were pre-treated with hypoxia for 30 min, 1–5 days later, tGCI was induced for 10 min (the number of rats in each group was 9, 9, 7, 6, and 7, respectively). Data are mean ± SD. *< 0.05 versus the tGCI control group.

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As shown in Fig. 1b, compared with the tGCI control group, exposing animals to hypoxia of 30, 60 or 120 min was able to offer protection against the neuronal damage by 10 min of tGCI performed 2 days later (< 0.05). The surviving cells numbers in the CA1 layer were 59.67 ± 18.05/mm, 50.38 ± 16.70/mm, and 43.67 ± 9.61/mm, respectively. Hypoxic duration of 30 min was most effective for reducing cell damage of CA1 subregion. However, longer periods of HPC (180 min) in surviving animals did not induce protection. The surviving cells number in the 180 min HPC group was 10.71 ± 6.55/mm.

To determine the hypoxia-ischemic interval, we selected 30 min hypoxia as the duration of pre-treatment for the subsequent study. As shown in Fig. 1a, ix and x (n = 9) and 1c, the maximum protective effect after 30 min of hypoxia was observed when 1 day was allowed to elapse between hypoxia and ischemia (the surviving cells number was 69.67 ± 8.16/mm). 2-, 3- and 4-day intervals between hypoxia and ischemia also led to statistically significant protection (the surviving cells numbers were 59.67 ± 18.05/mm, 48.43 ± 11.13/mm, and 50.50 ± 16.37/mm, respectively), while the 5-day interval did not differ from the tGCI control group (> 0.05).

Effects of hypoxic preconditioning on phosphorylation of Akt

To investigate the phosphorylation of Akt (Ser473) after tGCI with or without HPC in the maximum protection group (30 min of hypoxia followed by subsequent tGCI with a 1 day interval), western blot and immunohistochemistry were used. As shown in Fig. 2a and 2b of western blot analysis, in the tGCI control group, phospho-Akt significantly decreased to 47.17 ± 8.93% (p < 0.05, n = 4) of that in sham-operated animals immediately after ischemia, followed by a transient increase to 174.22 ± 31.99% 4 h after reperfusion, and then a persistent decrease to 63.02 ± 26.07% and 53.96 ± 10.85% 24 h and 48 h after reperfusion, respectively. In contrast, in the HPC group, phospho-Akt was dramatically increased after hypoxia and ischemia except for a transient decrease 0–4 h after reperfusion. No significant changes were observed regarding the total protein expression of Akt.

image

Figure 2.  Effect of hypoxic pre-conditioning on Akt phosphorylation in the CA1 subregion. (a) Representative images of western blot using either anti-phospho-Akt (Ser473) or anti-Akt antibody in ischemic and hypoxic preconditioned rats. (b) Quantitative analyses of phospho-Akt levels in the CA1 subregion in ischemic and hypoxic preconditioned rats. Data are expressed as percentage of value of sham-operated animals. Each bar represents the mean ± SD. *< 0.05 versus the sham-operated animals and #< 0.05 versus the transient global cerebral ischemia (tGCI) control group at the same time point (n = 4 in each group). (c) Immunohistochemistry for phospho-Akt in the rat brains of 24 h after 10 min of tGCI with hypoxic pre-conditioning. Phospho-Akt positive cells were observed mainly in cortex, striatum and hippocampus (i,iii,v). In the hippocampus, positive cells were distributed in the pyramidal cell layers (v–x). Positive phospho-Akt staining was located in the cytoplasm and dendrites (black arrow) (ii,iv). Scale bar: i,iii,v,vii,ix: 150 μm; ii,iv,vi,viii,x: 15 μm. (d) Quantitative analysis of immunoreactive cell counting of phospho-Akt in the CA1 subregion. Data are shown as mean ± SD. *< 0.05 versus the sham-operated animals and #< 0.05 versus the tGCI control group at the same time point (n = 6 in each group). (e) Representative photomicrographs show fluorescent double staining of phospho-Akt (green) and neuronal nuclei (NeuN; red), phospho-Akt (green) and microtubule associated protein-2 (MAP-2; red), and phospho-Akt (green) and glial fibrillary acidic protein (GFAP; red) in the CA1 subregion 24 h after 10 min of tGCI with hypoxic pre-conditioning. Phospho-Akt-positive cells were observed in the CA1 subregion (i,iv,vii). Neuron-specific nuclear protein immunohistochemistry shows neurons in the same view (ii). MAP-2-positive cells were observed in the CA1 subregion (v). Images with (i) and (ii) overlapped, and (iv) and (v) overlapped show that phospho-Akt-positive cells in the CA1 subregion expressed in neurons and located mainly in neuronal bodies and dendrites (iii,vi). GFAP-positive cells were observed in the CA1 subregion (viii). An image with (vii) and (viii) overlapped shows no colocalization of phospho-Akt and GFAP (ix). Scale bar: 150 μm. H, hypoxia only group; Is, tGCI control group; Pre, hypoxia pre-conditioning group.

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An immunohistochemistry study showed phospho-Akt positive cells were observed mainly in cortex, striatum and hippocampus (Fig. 2c). In the hippocampus, positive cells were distributed in the pyramidal cell layers (Fig. 2c, v–x). Positive phospho-Akt staining was located in the cytoplasm and dendrites (Fig. 2c, ii and iv). Cell counting showed slight immunoreactivity of phospho-Akt in hippocampal CA1 subregion in the Sham-operated group. Correspondingly to the results of western blot, in the tGCI control group, the immunostaining of phospho-Akt was significantly decreased after ischemia except for a transient increase 4 h after reperfusion and sustained the decreasing from 24 h to 7 days after reperfusion. In the HPC group, the immunostaining of phospho-Akt was decreased significantly from 0 h to 4 h after reperfusion but increased from 24 h to 48 h after reperfusion and then returned to basal expression level 7 days after reperfusion. Noteworthily, in the hypoxia only group, a significant increase of immunostaining of phospho-Akt was observed 24 h after hypoxia. The quantitative analysis was shown in Fig. 2d.

To examine which type of cells expressed phospho-Akt in ischemic brains either with or without HPC, immunofluorescent double staining was performed at 24 h after ischemia. No colocalization of phospho-Akt with Hoechst 33528 was observed (data not shown). We found that phospho-Akt was co-labeled with MAP-2 in ischemic brains with HPC. No colocalization of phospho-Akt with either NeuN or GFAP was observed. These indicated that phospho-Akt was expressed mainly in neuronal cell bodies and dendrites, instead of nuclei of neurons or astrocytes (Fig. 2e). In both Sham-operated and ischemic brains, the results of double-staining for phospho-Akt were similar except that the numbers of neurons expressing phospho-Akt were substantially fewer than those in the HPC brains (data not shown).

Effect of hypoxic pre-conditioning on phosphorylation of FoxOs

As phospho-Akt functions through phosphorylation and inhibition of FoxOs (Brunet et al. 1999), we further examined the phosphorylation of FoxOs (including FKHR, FKHRL1 and AFX) after ischemia with or without HPC. As shown in Fig. 3, the changes of phospho-FoxOs resembled those of phospho-Akt described above in western blot analysis. In the tGCI control group, phospho-FoxOs were significantly decreased after ischemia except for a transient increase 4 h after reperfusion. In contrast, in the HPC group, phospho-FoxOs was dramatically increased after ischemia except for a transient decrease 0–4 h after reperfusion. In the hypoxia only group, a significant increase of phospho-FoxOs was observed 24 h after hypoxia. No significant change was observed regarding the protein expression of FoxOs.

image

Figure 3.  Effect of hypoxic pre-conditioning on FoxOs phosphorylation in the CA1 subregion. (a) Western blot analysis for phospho-FKHR (Ser256) and FKHR in ischemic and hypoxic pre-conditioned rats. (b) Western blot analysis for phospho-FKHRL1 (Ser253) and FKHRL1 in ischemic and hypoxic pre-conditioned rats. (c) Western blot analysis for phospho-AFX (Ser197) and AFX in ischemic and hypoxic pre-conditioned rats. Images of western blot are shown on the left side. Quantitative analyses of phospho-FoxOs levels in the CA1 subregion are shown on the right side. Data are expressed as percentage of value of sham-operated animals. Each bar represents the mean ± SD. *< 0.05 versus the sham-operated animals and #< 0.05 versus the transient global cerebral ischemia control group at the same time point (n = 4 in each group).

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Correspondingly to the results of western blot, in the tGCI control group, the immunostaining of phospho-FoxOs were decreased immediately after ischemia, then increased transiently 4 h after reperfusion and sustained the decreasing from 24 h to 7 days after reperfusion. In the HPC group, the immunostaining of phospho-FoxOs were decreased significantly from 0 h to 4 h after reperfusion but increased from 24 h to 48 h after reperfusion, and then returned to basal expression level 7 days after reperfusion. In the hypoxia only group, we noted a significant increase of immunostaining of phospho-FoxOs 24 h after hypoxia. The quantitative analysis was shown in Fig. 4a–c.

image

Figure 4.  Effect of hypoxic pre-conditioning on FoxOs phosphorylation in the CA1 subregion. (a) Quantitative analysis of densitometric measurement phospho-FKHR in the CA1 subregion. (b) Quantitative analysis of immunoreactive cell counting of phospho-FKHRL1 in the CA1 subregion. (c) Quantitative analysis of immunoreactive cell counting of phospho-AFX in the CA1 subregion. Data are shown as mean ± SD. *< 0.05 versus the sham-operated animals and #< 0.05 versus the transient global cerebral ischemia control group at the same time point (n = 6 in each group). (d) Representative photomicrographs show fluorescent double staining of phospho-FKHR (green) and Hoechst 33528 (blue), phospho-FKHR (green) and neuronal nuclei (NeuN; red), phospho-FKHR (green) and microtubule associated protein-2 (MAP-2; red), and phospho-FKHR (green) and glial fibrillary acidic protein (GFAP; red) in the CA1 subregion 24 h after ischemia with hypoxic pre-conditioning. Phospho-FKHR-positive cells were observed in the CA1 subregion (i,iv,vii,x). Hoechst 33528 staining shows nuclei in the same view (ii). An image with (i) and (ii) overlapped shows colocalization of phospho-FKHR and Hoechst 33528 (iii). NeuN-positive cells and MAP-2-positive cells were observed in the CA1 subregion (v,viii). Images with (iv) and (v) overlapped, and (vii) and (viii) overlapped show that phospho-FKHR-positive cells in the CA1 subregion expressed in neurons and located mainly in nuclei (vi,ix). GFAP-positive cells were observed in the CA1 subregion (xi). An image with (x) and (xi) overlapped shows no colocalization of phospho-FKHR and GFAP (xii). Scale bar = 150 μm.

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To examine which type of cells expressed phospho-FKHR in ischemic brains either with or without HPC, immunofluorescent double staining was performed at 24 h after ischemia. We found that phospho-FKHR was co-labeled mainly with Hoechst 33528 and NeuN and slightly with MAP-2 in ischemic brains with HPC. No colocalization of phospho-FKHR with GFAP was observed. These indicated that phospho-FKHR was expressed mainly in nuclei, especially in the nuclei of neurons, instead of astrocytes (Fig. 4d). In both Sham-operated and ischemic brains, the results of double-staining for phospho-FKHR were similar except that the numbers of neurons expressing phospho-FKHR were substantially fewer than those in the HPC brains (data not shown).

Effects of LY294002 on phosphorylation levels of Akt and FoxOs and ischemic tolerance

To further confirm the role of Akt activation and FoxOs inactivation in induction of IT, we administrated LY294002, a PI3K inhibitor, before HPC or ischemia, examined the expression of phospho-Akt and phospho-FoxOs and evaluated the neuronal damage of the CA1 pyramidal cells. Interestingly, the increase of phospho-Akt and phospho-FoxOs 24 h after tGCI in the CA1 subregion with 30 min of hypoxia was not found in the LY294002-pretreated animals, which was different from what was observed in the vehicle-pretreated animals or the HPC animals. Also, compared with tGCI animals, phospho-Akt and phospho-FoxOs 24 h after tGCI with LY294002 pretreatment were decreased (n = 4 in each group, < 0.05, Figs 5a, b and 6). Delayed neuronal death was evaluated on day 7 after ischemia with or without LY294002 pre-treatment. Compared with sham-operated animals (n = 6, Fig. 5c, i and ii), treatment with LY294002 in sham-operated animals produced no neurotoxic effects (n = 6, Fig. 5c, iii and iv). Compared with HPC group (n = 9, Fig. 5c, v and vi), infusion of vehicle before HPC did not blocked the neuroprotective effect of pre-conditioning (n = 6, Fig. 5c, vii and viii), however, the neuroprotection offered by HPC was abolished in the LY294002-pretreated animals (n = 8, Fig. 5c, ix and x).

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Figure 5.  Effect of LY294002 treatment on Akt phosphorylation and induction of ischemic tolerance in the CA1 subregion. (a) Western blot analysis for phospho-Akt in the CA1 subregion. (b) Quantitation of phospho-Akt in the CA1 subregion. Data are expressed as percentage of value of sham-operated animals. Each bar represents the mean ± SD. *< 0.05 versus the sham-operated animals, #< 0.05 versus the hypoxic preconditioning (HPC) + Is group and ##< 0.05 versus the transient global cerebral ischemia (tGCI) control group (n = 4 in each group). (c) Photomicrograph of sections showing Cresyl violet staining of the hippocampus. Sham group (i,ii; n = 6), Sham + LY group, infusion with LY294002 without hypoxia or ischemia (iii,iv; n = 6), HPC + Is group (v,vi;, n = 9), vehicle + HPC + Is group, 10 min of tGCI with vehicle infusion before HPC (vii,viii; n = 6), LY + HPC + Is group, 10 min of tGCI with LY294002 infusion before HPC (ix,x; n = 8). Scale bar: i,iii,v,vii,ix: 250 μm, ii,iv,vi,viii,x: 25 μm. (d) Quantification of surviving cell in the CA1 subregion. Data are mean ± SD. *< 0.05 versus the sham-operated animals, and #< 0.05 versus the HPC + Is group. LY, LY294002.

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image

Figure 6.  Effect of LY294002 treatment on FoxOs phosphorylation in the CA1 subregion. (a) Western blot analysis for phospho-FKHR in the CA1 subregion. (b) Western blot analysis for phospho-FKHRL1 in the CA1 subregion. (c) Western blot analysis for phospho-AFX in the CA1 subregion. Data are expressed as percentage of value of sham-operated animals. Each bar represents the mean ± SD. *< 0.05 versus the sham-operated animals, #< 0.05 versus the hypoxic pre-conditioning (HPC) + Is group and ##< 0.05 versus the transient global cerebral ischemia control group (n = 4 in each group).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Induction of IT after hypoxia has already been reported in vitro and in vivo. However, in the latter case, this kind of tolerance had been described against global ischemia in the neonate or focal ischemia in the adult brain. To our knowledge, this is the first study to examine HPC of tGCI in adult rats. In addition, this study reported the role of Akt/FoxO signaling pathway in IT. The major findings of our study were as follows: (i) the maximum neuroprotection against tGCI was observed with 30 min of hypoxia and 1 day interval between hypoxia and ischemia, (ii) HPC induced a significant increase of phospho-Akt and phospho-FoxOs after ischemia except for a transient decrease, and (iii) inhibition of phospho-Akt and phospho-FoxOs reduced the protective effect of HPC, which provided evidence for a role of Akt/FoxO signaling pathway in IT.

Several types of stress can induce IT, such as brief global or focal ischemia, hyperthermia, and spreading depression (Kirino 2002; Blanco et al. 2006). These types of pre-conditioning can never been employed in clinical setting because of safety concerns. Our histopathologic analysis indicated that a short period of HPC was non-injurious. Up to 120 min hypoxia, there was no evidence of delayed cellular injury of the CA1 pyramidal cells that is highly vulnerable to global hypoxic ischemic injury.

The duration of hypoxia may influence the outcome of ischemia. Our results show that reduction of the HPC duration to 30 min is still enough to decrease the cell damage. The duration of hypoxia described in the literature is approximately 1–5 h (Bernaudin et al. 2002; Prass et al. 2003), which is longer than our shortest hypoxic duration (30 min). Our results show that 30 min of hypoxia exposition is enough to induce tolerance, and that this neuroprotection cannot be improved by increasing the duration of hypoxia. Indeed, in our model, pre-treated for 180 min hypoxia, half of the animals died, and the surviving animals showed cell damage in the CA1 sector after tGCI.

There are two temporally distinct types of IT afforded by sublethal pre-treatment – immediate and delayed tolerance. IT found in the brain is usually of the delayed type (Kirino 2002). In animal models, Kirino et al. (1991) found that the protection of IT once induced was believed to last for a few days and to wane gradually until it disappeared. Zhang et al. (2008) discovered that a repetitive focal ischemic pre-conditioning stimulus could be titrated to delayed tolerance when the interval between the pre-conditioning and test middle cerebral artery occlusion (MCAO) was 1, 2, 3, or 4 days, but that the maximal protection was seen with a 3-day interval. In clinical practice, if a stroke occurs within the same vascular territory during a limited time window (1–7 days) after an appropriate transient ischemic attack, the stroke may be less severe and the outcome may be better than otherwise (Schaller 2005). In our present study, an interval of 1–4 days could lead to IT, but 5 days was too long to induce tolerance, and the maximal protection was observed on day 1 after pre-conditioning. These results suggest that the time intervals between pre-treatment and the subsequent severe ischemia influence tolerance induction. It may be because of that the delayed tolerance requires a time window to synthesize the necessary proteins and activate genes (Barone et al. 1998).

As HPC is non-invasive, simple to perform and reproductive for IT, our model could be very useful for further study on the mechanisms of IT in the adult brain. Despite extensive research, the protective mechanisms of HPC are still not well understood. The survival or death of cells depends on the balance between pro-survival and pro-apoptosis signaling pathways. Under pathological conditions, such as ischemia, this balance may be destroyed, which leads to apoptosis. However, in IT, a new balance may be established.

Akt pathway is an important pro-survival signaling pathway. Changes in the phospho-Akt have been reported after brain ischemia (Osuka et al. 2004; Endo et al. 2006). We tested the possibility that the HPC would change the activation of Akt after ischemia. The present results demonstrated that phospho-Akt was transiently higher in the non-preconditioned rats than in the pre-conditioned ones (4 h after reperfusion). Ischemia/reperfusion has been reported to produce reactive oxygen species leading to severe oxidative stress (Morita-Fujimura et al. 2001; Kim et al. 2002). A previous report has shown that ischemic pre-conditioning results in an increase in the activity of superoxide dismutase, an anti-oxidant enzyme (Toyoda et al. 1997). These finding suggest that the oxidative stress is attenuated after ischemia in the pre-conditioned rats because of the preconditioning-induced increased activity of superoxide dismutase. As phospho-Akt in neurons increases after exposure to hydrogen peroxide in a dose-dependent manner (Crossthwaite et al. 2002), the present finding that phospho-Akt was transiently higher in the non-preconditioned rats than in the preconditioned ones may be attributed to the preconditioning-induced anti-oxidant effect. Although phospho-Akt was higher in the non-preconditioned rats than in the pre-conditioned ones early after ischemia, it rapidly decreased thereafter in the non-preconditioned ones. In contrast, such a rapid decrease in the activation level of Akt was prevented in pre-conditioned rats, and the activation level remained high in the pre-conditioned rats until 48 h after ischemia compared with non-preconditioned ones. These findings suggest that phospho-Akt induced after ischemia is inhibited in the pre-conditioned rats. Our result is consistent with reports from other studies using ischemic pre-conditioning (Yano et al. 2001; Yin et al. 2005). Our results also showed that phospho-Akt was increased 24 h after hypoxia, correlating with the development of IT. Interestingly, the decrease of phospho-Akt after tGCI with LY294002 pre-treatment was found. Accordingly, LY294002 treatment before HPC significantly prevented the neuroprotective action of pre-conditioning, with concomitant elimination of Akt activation. In addition, LY294002 treatment itself did not cause neuronal cell death. These results suggest that Akt activation after hypoxia and hypoxia-ischemia plays an important role in the establishment of IT. Unlike the rapid changes in phospho-Akt after brain ischemia, our results showed that total Akt protein level did not change following ischemia with or without HPC in CA1 subregion, which is consistent with almost all other studies (Yano et al. 2001; Yin et al. 2007). The results suggested that ischemia modulates neuronal death and HPC mediates IT through phosphorylation of Akt rather than through regulating its protein level.

Downstream targets for Akt that underlie neuroprotection have not been identified in the neurons. Akt prevents cell death through phosphorylating and inactivating downstream factors. The current study focused on phosphorylation of the FoxOs including FKHR, FKHRL1 and AFX after ischemia with or without HPC. It has been reported that phospho-FKHR decreases at 0, 30 and 60 min after reperfusion following 5 min of forebrain ischemia in gerbil models (Kawano et al. 2002). Additionally, Zhao et al. (2005) observed that phospho-FKHR decreased from 30 min to 48 h after focal ischemia in Sprague Dawley rats. Shioda et al. (2007b) reported that phospho-FKHR and phospho-FKHRL1 decreased from 2–6 h after 90 min of MCAO in mouse. These studies suggest that phospho-FoxOs such as FKHR and FKHRL1 decrease persistently after brain ischemia. Such results had a little distinction from ours. Our data demonstrated that phospho-FoxOs decreased after ischemia except for a transient increase. We believe that these different findings may be because of the difference in animal age and the model of ischemia. Ischemia/reperfusion causes inactivation of Akt, thereby decreasing phospho-FoxOs. The ultimate persistent dephosphorylation of FoxOs may contribute to the ischemic induced neuronal death.

The effect of HPC on phospho-FoxOs is first documented here. Our data showed increased phospho-FoxOs by increased Akt activity following tGCI likely mediated the neuroprotection induced by HPC. By applying another approach different from HPC, pre- and post-treatments with bis (1-oxy-2-pyridinethiolato) oxovanadium (IV), Shioda et al. (2007b) reported that phospho-FoxOs played an important role in protecting neurons after transient MCAO in mouse. Although using different treatments, it is possible that like bis (1-oxy-2-pyridinethiolato) oxovanadium (IV), HPC induced neuroprotection through inhibiting reduced Akt and FoxOs phosphorylation after brain ischemia. Further, the PI3K inhibitor, LY294002 prevented phosphorylation of Akt and FoxOs 24 h after ischemia/reperfusion with HPC, followed by increased neuronal damage, which meant that LY294002 blocked the protective effect of HPC. These results suggest that activation of Akt leads to the inactivation of FoxOs that may mediate IT after HPC. Although the precise mechanism underlying inactivation of FoxOs leading to IT after HPC is unclear, phospho-FoxOs likely induce cell survival by down-regulating apoptosis-inducing factors such as Bim and Fas-ligand. Bim is one of Bcl-2 members, and causes apoptosis in various cell types. Bim is also expressed in neuronal cells (O’Reilly et al. 2000). Fas is a member of the tumor necrosis factor receptor family, and its ligand, Fas-ligand, plays important roles in apoptosis (Nagata 1997). Activation of Fas leads to formation of a death-inducing signaling complex composed of the Fas-associated death domain and pro-caspase 8. Pro-caspase 8 is proteolytically cleaved and consequently activates caspase pathways and thereby cells are led to apoptosis. Recent studies reported that Bim (Fukunaga et al. 2005) and Fas-ligand (Fukunaga et al. 2005; Shioda et al. 2007a,b) expression significantly increased after brain ischemia/reperfusion accompanyed by dephosphorylation of FKHR. Therefore, it is possible that the phosphorylation of FoxOs induced by HPC via activation of Akt can block the expression of Bim and Fas-ligand, leading to cell survival. Further experiments will be required to investigate this interesting possibility.

In summary, we present a strong evidence of neuroprotection against brain injury by tGCI in adult rats using a short period of hypoxia. Activation of the Akt/FoxO signaling pathway is associated with the HPC in adult rats by inhibition of dephosphorylation of phospho-Akt and phospho-FoxOs. To understand the mechanisms induced by hypoxia that lead to neuroprotection against ischemia would be very important for identifying potential novel therapeutic targets in the field of ischemic stroke.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Jimin Zhang (Joint Program of Molecular Biosciences, Rutgers University & University of Medicine & Dentistry of New Jersey, USA) for helpful comments. This work was supported by Natural Science Foundation of Guangdong, China (Project 8151018201000035).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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