Background

Recent studies suggest that the novel neuropeptide orexin-A may play an essential role during neuronal damage. However, the function of orexin-A during brain ischemia remains unclear. Recently, hypoxia-inducible factor-1α (HIF-1α) was shown to be activated by orexin-A. The aim of the current study is to test the hypothesis that administration of exogenous orexin-A can attenuate ischemia-reperfusion injury through the facilitation of HIF-1α expression.

Methods

Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion for 120 min. Rats were treated with different doses of orexin-A or vehicle before the ischemia and at the onset of reperfusion. To investigate the action of HIF-1α in the neuroprotective effects of orexin-A, the HIF-1α inhibitor YC-1 was used alone or combined with orexin-A. Neurologic deficit scores and infarct volume were assessed. Brains were harvested for immunohistochemical staining and western blot analysis.

Results

Orexin-A significantly ameliorated neurologic deficit scores and reduced infarct volume after cerebral ischemia reperfusion. Administration of 30 μg/kg orexin-A showed optimal neuroprotective effects. This effect was still present 7 days after reperfusion. Furthermore, orexin-A decreased the number of apoptotic cells and significantly enhanced HIF-1α expression after cerebral ischemia reperfusion. Moreover, the facilitation of HIF-1α expression was accompanied with inhibition of von Hippel-Lindau expression. Administration of HIF-1α inhibitor suppressed the increase of HIF-1α and reversed the neuroprotective effects of orexin-A.

Conclusions

Orexin-A has a neuroprotective effect against cerebral ischemia-reperfusion injury. These effects may be mediated through the HIF-1α pathway.

  • Reductions in the endogenous neuropeptide orexin-A have been implicated in the pathophysiology of stroke.

  • Intravenous orexin-A was neuroprotective in a rat model of transient cerebral ischemia by a mechanism involving hypoxia-inducible factor-1α, representing a novel potential therapy for stroke and neuroprotection.

OREXINS are neuropeptides that were initially characterized as potent stimulants of food intake,1but they also have various physiologic effects, including energy homeostasis, neuroendocrine and autonomic nervous system functions, pain perception, and sleep-wake cycle control.2,3For example, Peyron et al.  4discovered that reduced production of orexin peptides occurred in patients with narcolepsy. In addition, deletion of the orexin gene in mice produces a condition similar to human narcolepsy.5Recently, we demonstrated that orexinergic signals are involved in regulation of the anesthesia-awake cycle.6,7 

It is also well known that various physiologies, including sleep patterns,8,9energy homeostasis,10autonomic functions, and neuroendocrine functions11,12are often abnormal in patients with stroke. These phenomena inspired us to speculate that there is a correlation between the orexin system and cerebral ischemic injury.

Recently, a series of clinical and empirical studies have provided solid evidence supporting our speculation. A new published clinical study observed a significant transient decline in orexin-A concentrations at the onset of delayed ischemic neuronal deficits resulting from symptomatic vasospasm in patients with subarachnoid hemorrhage.13Nishino et al.  8also found that patients with cerebral infarction showed a persistent decrease of cerebrospinal orexin-A concentrations. In addition, a number of animal studies have shown that global or focal cerebral ischemia induces increased expression of orexin 1 receptors (OX1R) in the brain, which correlated with decreases of orexin-A in cerebrospinal fluid.14–16The accumulated evidence indicates that orexins, especially orexin-A, may play a pivotal role in neuronal cell death in cerebrovascular diseases and ischemia-reperfusion injury. Nevertheless, the direct effect of orexin-A on brain ischemic injury is poorly understood.

Hypoxia-inducible factor-1α (HIF-1α) is an important transcription factor involved in ischemic and hypoxic conditions.17Recently, an in vitro  experiment revealed that orexin-A can activate expression of HIF-1α by inhibiting von Hippel-Lindau (vHL) expression.18Thus, the current study was designed to test the hypothesis that administration of exogenous orexin-A can induce neuroprotective effects—as mediated by facilitation of HIF-1α expression—against cerebral ischemia-reperfusion injury in rats.

Animals

All procedures were carried out according to protocols approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University (Xi'an, Shaanxi, China) and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals . Adult male Sprague-Dawley rats (280–320g) obtained from the laboratory animal center of the Fourth Military Medical University were used in this study.

Experimental Protocol

Part I: Neuroprotective Effects of Orexin-A.

To investigate the neuroprotective effects of orexin-A (Phoenix Biotech, Mississauga, Ontario, Canada), 48 rats were randomly assigned to one of six groups (n = 8): sham, control, OXA-3, OXA-10, OXA-30, and OXA-100 groups. At the onset of reperfusion, rats were randomized to receive either vehicle (phosphate buffered saline [PBS]) or orexin-A (3, 10, 30, or 100 μg/kg, diluted in PBS) by intravenous injection (0.5 ml). At 24 and 72 h after reperfusion, neurologic scores were evaluated in a masked fashion. At 72 h after reperfusion, rats were sacrificed for infarct volume measurement.

Part II: Sustained Neuroprotective Effects of Orexin-A.

To investigate the sustained neuroprotective effects of orexin-A as well as differences between preischemic and postischemic administration, 32 rats were randomly assigned to one of four groups (n = 8): sham, control, OXA-pre, and OXA-post groups. For the OXA-pre group, orexin-A (30 μg/kg, diluted in PBS) was administered by intravenous injection (0.5 ml) 10 min before ischemia. For the OXA-post group, orexin-A (30 μg/kg, diluted in PBS) was administered by intravenous injection (0.5 ml) at the onset of reperfusion in rats. For sham and control groups, PBS (0.5 ml) was administered intravenously 10 min before ischemia and at the onset of reperfusion in rats. At 3 and 7 days after reperfusion, neurologic scores were evaluated in a masked fashion and rats were sacrificed for infarct volume assessment.

Part III: HIF-1α and vHL Regulation after Orexin-A.

To explore whether the neuroprotective effects of orexin-A are mediated by the HIF-1α pathway, the expression of HIF-1α and vHL were observed after cerebral ischemia reperfusion with or without orexin-A treatment. According to the results from the first part of this experiment, 30 μg/kg orexin-A was used in the treatment group. Rats were randomly assigned to one of three groups (n = 36): sham, control, and orexin-A treatment groups. At the onset of reperfusion, rats were randomized to receive either vehicle (PBS) or orexin-A (30 μg/kg diluted in PBS) by intravenous injection (0.5 ml). Immunohistochemistry and western blot analysis were performed 2, 24, and 72 h after reperfusion (n = 12 at each time point).

Part IV: Effects of HIF-1α Inhibitor YC-1 on Neuroprotective Properties of Orexin-A and HIF-1α Protein Expression.

To demonstrate the involvement of the HIF-1α pathway in the neuroprotective effects of orexin-A, 40 rats were randomly assigned to one of five groups (n = 8): sham; control; YC-1(3-(50-hydroxymethyl-20-furyl)-1-benzylindazole, the inhibitor of HIF-1α; orexin-A; and orexin-A + YC-1. At the onset of reperfusion, rats were randomized to receive either vehicle (1% dimethyl sulfoxide), YC-1 (1 mg/kg diluted in 1% dimethyl sulfoxide PBS), orexin-A (30 μg/kg diluted in PBS), or orexin-A combined with YC-1 by intravenous injection (0.5 ml). At 24 and 72 h after reperfusion, neurologic scores were evaluated in a masked fashion. At 72 h after reperfusion, rats were sacrificed for infarct volume measurement.

Western blot analysis was used to check the effect of YC-1 on HIF-1α protein expression. Sixty rats were randomly assigned to one of five groups: sham, control, YC-1, orexin-A, and orexin-A + YC-1 group. At 24 and 72 h after reperfusion, rats (n = 6 at each time point) were sacrificed and the hippocampus and penumbral cortex of the right hemisphere was dissected for western blot analysis.

Transient Middle Cerebral Artery Occlusion Model

The transient middle cerebral artery occlusion (MCAO) model was performed as previously described.19After an overnight fast, animals were anesthetized by intraperitoneal injection of 40–50 mg/kg sodium pentobarbital, 2% (v/v), for all surgical procedures. Transient focal cerebral ischemia was produced by intraluminal suture occlusion of the right middle cerebral artery using a 3-0 monofilament nylon suture (Ethicon, Inc., Osaka, Japan). After 2 h of MCAO, the suture was carefully removed from the internal carotid artery. The neck incision was closed and rats were allowed to recover. Sham-operated rats underwent the same surgical procedure except that the suture was not inserted into the internal carotid artery.

Regional cerebral blood flow was monitored using a flexible optical fiber probe attached to the skull over the ipsilateral parietal cortex at one point (1 mm posterior and 5 mm lateral to the bregma) by laser Doppler flowmetry (PeriFlux system 5000; Perimed AB, Stockholm, Sweden). Rats in which ipsilateral blood flow was not reduced to less than 20% of the baseline after placement of the intraluminal filament and whose cerebral blood flow signal was not rapidly restored during reperfusion were excluded from subsequent experiments. Cranial temperature was maintained at 36.8-37.5°C with a heating pad. In a separate experiment, physiologic parameters (cranial temperature, arterial pH, PaCO2, PaO2, glucose) were monitored and analyzed in six additional rats. Arterial blood samples were taken 3 min before ischemia (baseline), 60 min after ischemia, and 30 min after reperfusion for gases and plasma glucose measurements.

Neurologic Scores

Neurologic scores were evaluated 24 and 72 h after reperfusion by using a scoring system reported by Garcia et al.  20in a masked fashion. This system consisted of the following six tests: (1) spontaneous activity, (2) symmetry in the movement of four limbs, (3) forepaw outstretching, (4) climbing, (5) body proprioception, and (6) response to vibrissae touch. The score given to each rat at the completion of the evaluation was the summation of all six individual test scores. Minimum neurologic score was 3; maximum was 18.

Infarct Volume Measurement

To compare infarct sizes in the ischemia-treated groups, infarct volume was measured (at 72 h after reperfusion; n = 8 per group) using 2,3,5-triphenyltetrazolium chloride staining (Sigma-Aldrich, St. Louis, MO) as described in our previous report.19Infarct and total hemispheric areas of sections were traced from slices taken at 2-mm intervals and measured using an image analysis system (Adobe Photoshop 8.0, Adobe Systems Incorporated, San Jose, CA). To compensate for the effect of brain edema, corrected infarct volumes were calculated as previously described using the following equation: corrected infarct area = measured infarct area ×{1 −[(ipsilateral hemisphere area − contralateral hemisphere area)/contralateral hemisphere]}.21Infarct volumes were expressed as percentages of contralateral hemispheric volumes.

Specimen Preparation

For immunohistochemical analysis, animals (n = 6 at each time point) were anesthetized with an overdose of 2% (v/v) sodium pentobarbital and perfusion-fixed via  the left ventricle as previously described at 2, 24, and 72 h after MCAO.22,23Animals were first perfused with 200 ml 0.1 M PBS and then with 400 ml paraformaldehyde, 4% (w/v), in 0.1 M PBS (pH 7.4). Brains were removed and postfixed in 4% (w/v) paraformaldehyde in 0.1 M PBS. Brain blocks were embedded in paraffin and cut into 3-μm coronal sections. Sections were used for immunohistochemical staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end label (TUNEL) staining.

For western blot analysis, rats (n = 6 at each time point) were sacrificed at designated time intervals. Brains were removed and the hippocampus and penumbral cortex of the right hemisphere was dissected according to well-established protocols in rodent models of MCAO.24Samples were frozen immediately on dry ice and stored at −80°C until ready for use.

Immunohistochemical Staining

Sections were immunostained for rabbit anti-HIF-1α antibody (ab51608, 1:200; Abcam, Cambridge, MA) and goat anti-vHL protein antibody (ab77262, 1:300; Abcam). A microwave oven was used to dewax paraffin sections for 10 min in 0.01 M citrate buffer solution (pH 6.0). Sections were incubated in 3% (v/v) H2O2for 10 min, to prevent reaction with endogenous peroxidases, and 30-min incubation with 10% (v/v) normal goat serum or rabbit serum at room temperature. Sections were then incubated with the primary antibodies overnight at 4°C.

For HIF-1α staining, after washing in PBS, sections were incubated with peroxidase anti-rabbit IgG secondary antibody solution (1:500; Vector Laboratories, Burlingame, CA) in a humidified chamber at room temperature for 1 h. For vHL staining, sections were washed with PBS and incubated with biotinylated anti-goat IgG secondary antibody solution (1:800; Vector Laboratories) in a humidified chamber at room temperature for 1 h. Sections were then incubated with streptavidin-biotin complex in a humidified chamber for 30 min at room temperature. All sections were developed with diaminobenzidine for 5 min at room temperature. Sections were counterstained with hematoxylin, dehydrated with a series of ethanol solutions, coverslipped, and observed under a microscope (BX51; Olympus Corporation, Tokyo, Japan). Application of control serum instead of the primary antibody on other sections of the same brain provided a negative control. No immunostaining was observed in these sections.

Integrated optical density, a parameter representing expression of HIF-1α and vHL proteins in the hippocampus CA1 region and penumbral cortex, was measured quantitatively via  digital image analysis (Image-Pro Plus 5.0; Media Cybernetics, Inc., Bethesda, MD) in four fields per slide. The average value of the integrated optical density in six animals in each group was calculated.

TUNEL Staining and Quantification of Apoptosis

Apoptotic cells were detected by TUNEL. Fragmented DNA in cell nuclei was labeled using an In Situ  Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany) according to manufacturer instructions. Sections were stained by TUNEL and then were counterstained with hematoxylin. A neuron with a nucleus stained dark brown was considered a positive cell. By counterstaining with hematoxylin (a blue dye), viable neurons were also observed. TUNEL-positive cell numbers were counted in a masked fashion in four fields in the penumbral cortex of the right hemisphere at high-power microscopic magnification (×400) and expressed as the number of positive cells per high-power field. Data were analyzed from six animals per group at each time point.

Western Blot Analysis

Methods for western blot analysis have been described previously.25Tissue samples were homogenized using a whole protein extract kit (Millipore Corporation, Billerica, MA) with a glass tissue grinder. Protein content was determined using the BCA protein assay kit (Millipore Corporation). Equal amounts of protein per lane (50 μg) were loaded onto an 8% (v/v) polyacrylamide gel and separated by electrophoresis at 80 V for 30 min and 120 V for 70 min. Proteins were then transferred to nitrocellulose (Bio-Rad Laboratories, Inc., Hercules, CA) at 20 V for 50 min and the membrane was blocked with 10% (w/v) nonfat dry milk and 0.5% (v/v) Tween-20 in Tris-buffered saline. Nitrocellulose was incubated with two different antibodies overnight at 4°C, namely rabbit anti-HIF-1α antibody (ab51608, 1:1,000; Abcam) and goat anti-vHL protein antibody (ab77262, 1:1,000; Abcam). The membrane was treated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG or anti-goat IgG, 1:2000, Vector Laboratories) for 45 min at 37°C. The specific protein bands were visualized using the standard chemical luminescence method (ECL Kit; Amersham Pharmacia Biotech, Piscataway, NJ). Image analysis was accomplished with the assistance of computerized analysis software (Bio-Rad Laboratories, Inc.). Protein concentration was analyzed densitometrically and corrected with values determined on anti-β-actin blots. The density of proteins was expressed as relative values.

Statistical Analysis

All data, except for neurologic scores, are expressed as mean ± SD. Physiologic parameters were analyzed by repeated-measures ANOVA. Infarct volumes among different groups were compared by one-way ANOVA. When ANOVA showed significant differences, the post hoc  multiple-comparison Tukey test was applied. For the analysis of neuron apoptosis and interest protein expression quantification, two-way ANOVA was applied to determine if there was interaction between the two main effects (treatment and time). In case of a significant interaction, the post hoc  multiple comparison was performed to compare the difference among treatment groups at each time point. Otherwise, global conclusions were drawn based on the main effect. Neurologic scores were expressed as median (range) and were compared by Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction. The nature of hypothesis testing was two-tailed test, with P < 0.05 accepted as statistically significant. All statistical analyses were performed with SPSS 12.0 for Windows (SPSS, Inc., Chicago, IL).

Physiologic Parameters and Regional Cerebral Blood Flow

No statistical significance was noted among different time points for any of the physiologic parameters including cranial temperature, blood gas, and glucose concentrations (table 1). Physiologic parameters remained in the normal range during the experimental period. Monitoring of regional cerebral blood flow demonstrated successful MCAO (fig. 1).

Table 1.  Physiological Parameters, Mean ± SD (N = 6)

Table 1.  Physiological Parameters, Mean ± SD (N = 6)
Table 1.  Physiological Parameters, Mean ± SD (N = 6)

Fig. 1.  Regional cerebral blood flow (rCBF) was monitored 5 min before transient middle cerebral artery occlusion (MCAO), at 5, 60, and 115 min during MCAO, and 5 min after MCAO. Regional cerebral blood flow monitoring demonstrated if the MCAO model was successful. CON = control; OXA-3 = 3 μg/kg orexin-A; OXA-10 = 10 μg/kg orexin-A; OXA-30 = 30 μg/kg orexin-A; OXA-100 = 100 μg/kg orexin-A.

Fig. 1.  Regional cerebral blood flow (rCBF) was monitored 5 min before transient middle cerebral artery occlusion (MCAO), at 5, 60, and 115 min during MCAO, and 5 min after MCAO. Regional cerebral blood flow monitoring demonstrated if the MCAO model was successful. CON = control; OXA-3 = 3 μg/kg orexin-A; OXA-10 = 10 μg/kg orexin-A; OXA-30 = 30 μg/kg orexin-A; OXA-100 = 100 μg/kg orexin-A.

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Effects of Orexin-A-induced Neuroprotection on Neurologic Scores and Infarct Volume

Neurologic scores evaluated at 24 and 72 h after reperfusion in the sham, control, and the orexin-A-treated groups are shown in figure 2. Orexin-A significantly attenuated early neurologic deficits after MCAO (P < 0.05).

Fig. 2.  Neurologic scores and infarct volume after 120 min of transient middle cerebral artery occlusion in sham, control (CON), and orexin-A treatment groups. Orexin-A was administered at the onset of reperfusion by intravenous injection. Neurologic scores are presented as median (range); data for infarct volume are expressed as mean ± SD. Neurologic scores were evaluated at 24 h (A ) and at 72 h (B ) after reperfusion using the Garcia scoring system. (C ) Representative 2,3,5-triphenyltetrazolium chloride staining of the cerebral infarct in the rat brain at 72 h after reperfusion. (D ) Statistical analysis of the percentage of infarct volume was determined for each study group (n = 8). OXA-3 = 3 μg/kg orexin-A; OXA-10 = 10 μg/kg orexin-A; OXA-30 = 30 μg/kg orexin-A; OXA-100 = 100 μg/kg orexin-A. *P < 0.05 versus  control. **P < 0.01 versus  control.

Fig. 2.  Neurologic scores and infarct volume after 120 min of transient middle cerebral artery occlusion in sham, control (CON), and orexin-A treatment groups. Orexin-A was administered at the onset of reperfusion by intravenous injection. Neurologic scores are presented as median (range); data for infarct volume are expressed as mean ± SD. Neurologic scores were evaluated at 24 h (A ) and at 72 h (B ) after reperfusion using the Garcia scoring system. (C ) Representative 2,3,5-triphenyltetrazolium chloride staining of the cerebral infarct in the rat brain at 72 h after reperfusion. (D ) Statistical analysis of the percentage of infarct volume was determined for each study group (n = 8). OXA-3 = 3 μg/kg orexin-A; OXA-10 = 10 μg/kg orexin-A; OXA-30 = 30 μg/kg orexin-A; OXA-100 = 100 μg/kg orexin-A. *P < 0.05 versus  control. **P < 0.01 versus  control.

Close modal

Representative coronal brain sections stained with 2% (w/v) 2,3,5-triphenyltetrazolium chloride from the six groups are shown in figure 2C. Mean infarction volume percentage in the control group was 50.4%, whereas treatment with orexin-A reduced brain infarct volume in a dose-dependent manner from 10 to 30 μg/kg. However, when the dose of orexin-A was increased to 100 μg/kg, a slight increase in infarct volume was observed when compared with the 30 μg/kg dose. These results demonstrate that orexin-A exerts greater neuroprotection at a therapeutic dose of 30 μg/kg in rats (fig. 2D; P = 0.001).

Sustainable Neuroprotective Effects of Orexin-A

Neurologic scores evaluated at 3 and 7 days after reperfusion in the sham group, control group, OXA-pre, and OXA-post groups are shown in figure 3. Both administration schedules significantly attenuated neurologic deficits at 3 and 7 days after MCAO (P < 0.05).

Fig. 3.  Neurologic scores and infarct volume after 120 min of transient middle cerebral artery occlusion in sham, control (CON), and orexin-A treatment groups. Orexin-A was administered preischemia (OXA-pre) and postischemia (OXA-post). Neurologic scores are presented as median (range); data for infarct volume are expressed as mean ± SD. Neurologic scores were evaluated at 3 days (A ) and at 7 days (B ) after reperfusion using the Garcia scoring system. (C ) Representative 2,3,5-triphenyltetrazolium chloride staining of the cerebral infarct in the rat brain at 7 days after reperfusion. (D ) Statistical analysis of the percentage of infarct volume was determined for each study group (n = 8). *P < 0.05 versus  control.

Fig. 3.  Neurologic scores and infarct volume after 120 min of transient middle cerebral artery occlusion in sham, control (CON), and orexin-A treatment groups. Orexin-A was administered preischemia (OXA-pre) and postischemia (OXA-post). Neurologic scores are presented as median (range); data for infarct volume are expressed as mean ± SD. Neurologic scores were evaluated at 3 days (A ) and at 7 days (B ) after reperfusion using the Garcia scoring system. (C ) Representative 2,3,5-triphenyltetrazolium chloride staining of the cerebral infarct in the rat brain at 7 days after reperfusion. (D ) Statistical analysis of the percentage of infarct volume was determined for each study group (n = 8). *P < 0.05 versus  control.

Close modal

Representative coronal brain sections stained with 2% (w/v) 2,3,5-triphenyltetrazolium chloride from the four groups are shown in figure 3C. The mean infarction volume percentage in the control group was 44.7%, whereas treatment with orexin-A preischemia and postischemia reduced brain infarct volume (fig. 3D; OXA-pre, P = 0.003; OXA-post, P = 0.008). For neurologic scores and infarction volume, there was no significant difference between orexin-A groups regardless of administrative timing. These results demonstrate that orexin-A has sustainable neuroprotective effects regardless of administration timing (preischemia vs . postischemia).

TUNEL Staining

Ischemia-induced brain injury and the neuroprotective effects of orexin-A treatment were also assessed by observation of cell apoptosis in the brain with TUNEL staining. Figure 4Ashows TUNEL staining in the penumbral cortex of the right hemisphere. The bar graph (fig. 4B) shows quantitative analysis of the number of TUNEL-positive neurons. Apoptotic neurons appeared 2 h after reperfusion and increased at 24 and 72 h after reperfusion in the control group. The number of apoptotic neurons in the 30-μg/kg orexin-A treatment group was significantly lower than that in the control group at 24 h (P = 0.008) and 72 h (P = 0.001) after reperfusion. At 2 h after reperfusion, there was no significant difference in the number of apoptotic neurons between the control group and orexin-A treatment group.

Fig. 4.  (A ) Representative sections of nuclear DNA fragmentation assays performed by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining in the penumbral cortex in sham, control (CON) and 30 μg/kg orexin-A (OXA) treatment groups (n = 6). (B ) Quantitative analysis of the number of TUNEL-positive neurons in the penumbral cortex. Data are presented as mean ± SD. Scale bar, 10 μm. #P < 0.05 versus  sham. *P < 0.05 versus  control.

Fig. 4.  (A ) Representative sections of nuclear DNA fragmentation assays performed by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining in the penumbral cortex in sham, control (CON) and 30 μg/kg orexin-A (OXA) treatment groups (n = 6). (B ) Quantitative analysis of the number of TUNEL-positive neurons in the penumbral cortex. Data are presented as mean ± SD. Scale bar, 10 μm. #P < 0.05 versus  sham. *P < 0.05 versus  control.

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Expression of HIF-1α

Immunohistochemistry.

Immunohistochemical staining of HIF-1α in the hippocampus CA1 region and penumbral cortex are shown in figure 5and figure 6. At 2 h after reperfusion, a small number of HIF-1α-positive neurons were observed in the control group. Increased expression of HIF-1α was observed in sections from the control group 24 and 72 h after reperfusion. Orexin-A treatment (30 μg/kg) enhanced the staining of HIF-1α in the hippocampus CA1 region (24 h, P = 0.026; 72 h, P = 0.007) and penumbral cortex (24 and 72 h, P = 0.008) after reperfusion. However, there was no conspicuous distinction of HIF-1α staining between the control group and orexin-A group 2 h after reperfusion. There was no significant immunohistochemical staining for HIF-1α observed in the hippocampus CA1 region and the penumbral cortex in the sham group.

Fig. 5.  (A ) Representative sections of immunostaining for hypoxia-inducible factor-1α in the hippocampus CA1 region in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. (B ) Quantitative analysis of the integrated optical density (IOD). Data are presented as mean + SD. Scale bar, 20 μm. #P < 0.05 versus  sham. *P < 0.05 versus  control.

Fig. 5.  (A ) Representative sections of immunostaining for hypoxia-inducible factor-1α in the hippocampus CA1 region in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. (B ) Quantitative analysis of the integrated optical density (IOD). Data are presented as mean + SD. Scale bar, 20 μm. #P < 0.05 versus  sham. *P < 0.05 versus  control.

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Fig. 6.  (A ) Representative sections of immunostaining for hypoxia-inducible factor-1α in the penumbral cortex in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. (B ) Quantitative analysis of the integrated optical density (IOD). Data are presented as mean ± SD. Scale bar, 20 μm. #P < 0.05 versus  sham. *P < 0.05 versus  control.

Fig. 6.  (A ) Representative sections of immunostaining for hypoxia-inducible factor-1α in the penumbral cortex in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. (B ) Quantitative analysis of the integrated optical density (IOD). Data are presented as mean ± SD. Scale bar, 20 μm. #P < 0.05 versus  sham. *P < 0.05 versus  control.

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Western Blot Analysis.

Protein expression of HIF-1α in the hippocampus in the control group increased significantly at 2 h (P = 0.035) and peaked at 24–72 h (P < 0.001) after reperfusion when compared with the sham group. Orexin-A treatment (30 μg/kg) significantly increased the expression of HIF-1α at 24 h (P = 0.002) and 72 h (P < 0.001) after reperfusion when compared with the control group (fig. 7A).

Fig. 7.  Protein expression of hypoxia-inducible factor-1α (HIF-1α) in the hippocampus (A ) and penumbral cortex (B ) in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups were evaluated by Western blot analysis. Representative bands and corresponding β-actin bands (top ) and analysis of HIF-1α expression (bottom ) are shown for each study group. Data are presented as mean ± SD. #P < 0.05 versus  sham. *P < 0.05 versus  control.

Fig. 7.  Protein expression of hypoxia-inducible factor-1α (HIF-1α) in the hippocampus (A ) and penumbral cortex (B ) in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups were evaluated by Western blot analysis. Representative bands and corresponding β-actin bands (top ) and analysis of HIF-1α expression (bottom ) are shown for each study group. Data are presented as mean ± SD. #P < 0.05 versus  sham. *P < 0.05 versus  control.

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Similar HIF-1α protein expression was observed in the penumbral cortex. Orexin-A treatment increased HIF-1α expression at 24 h (P < 0.001) and 72 h (P = 0.003) after reperfusion (fig. 7B).

vHL Expression

Immunohistochemistry.

Immunohistochemical staining of vHL in the hippocampus CA1 region and penumbral cortex are shown in figure 8and figure 9. vHL expression in the control group did not show any significant changes when compared with the sham group. Orexin-A treatment (30 μg/kg) inhibited the increasing expression of vHL at 24 and 72 h after reperfusion in the hippocampus CA1 (P = 0.016) region and penumbral cortex (P = 0.002). However, there was no significant difference between the control group and orexin-A group at 2 h after reperfusion.

Fig. 8.  (A ) Representative sections of von Hippel-Lindau immunohistochemical staining in the hippocampus CA1 region in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. (B ) Quantitative analysis of the integrated optical density (IOD). Data are presented as mean ± SD. Scale bar, 20 μm. #P < 0.05 versus  sham. *P < 0.05 versus  control.

Fig. 8.  (A ) Representative sections of von Hippel-Lindau immunohistochemical staining in the hippocampus CA1 region in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. (B ) Quantitative analysis of the integrated optical density (IOD). Data are presented as mean ± SD. Scale bar, 20 μm. #P < 0.05 versus  sham. *P < 0.05 versus  control.

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Fig. 9.  (A ) Representative sections of von Hippel-Lindau immunohistochemical staining in the penumbral cortex in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. (B ) Quantitative analysis of the integrated optical density (IOD). Data are presented as mean ± SD. Scale bar, 20 μm. #P < 0.05 versus  sham, *P < 0.05 versus  control.

Fig. 9.  (A ) Representative sections of von Hippel-Lindau immunohistochemical staining in the penumbral cortex in sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. (B ) Quantitative analysis of the integrated optical density (IOD). Data are presented as mean ± SD. Scale bar, 20 μm. #P < 0.05 versus  sham, *P < 0.05 versus  control.

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Western Blot Analysis.

Immunohistochemical findings were confirmed by western blot analysis. The expression of vHL in the hippocampus in the control group did not show significant changes after ischemia reperfusion when compared with the sham group. vHL expression was significantly inhibited by 30 μg/kg orexin-A treatment at 24 h (P = 0.039) and at 72 h (P = 0.037) after reperfusion as compared with the control group (fig. 10A). Results from western blot analysis revealed that the penumbral cortex region and hippocampus had similar vHL expression patterns (fig. 10B; 24 h, P = 0.028; 72 h, P = 0.031).

Fig. 10.  Western blot analysis of von Hippel-Lindau expression in the hippocampus (A ) and in the penumbral cortex (B ) in the sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. Representative von Hippel-Lindau bands, corresponding β-actin bands (top ), and statistical results (bottom ) are shown for each study group (n = 6). Data are presented as mean ± SD. #P < 0.05 versus  sham, *P < 0.05 versus  control.

Fig. 10.  Western blot analysis of von Hippel-Lindau expression in the hippocampus (A ) and in the penumbral cortex (B ) in the sham, control (CON), and 30 μg/kg orexin-A (OXA) treatment groups. Representative von Hippel-Lindau bands, corresponding β-actin bands (top ), and statistical results (bottom ) are shown for each study group (n = 6). Data are presented as mean ± SD. #P < 0.05 versus  sham, *P < 0.05 versus  control.

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Effect of HIF-1α Inhibitor YC-1 on Neuroprotective Effects of Orexin-A

Neurologic scores evaluated at 24 and 72 h after reperfusion are shown in figure 11. In figures 11A and B, the neurologic score of orexin-A combined with YC-1 group was reduced as compared with orexin-A group. This result indicates that administration of orexin-A combined with YC-1 deteriorated rather than reduced the neurologic deficits when compared to OXA group.

Fig. 11.  Neurologic scores and infarct volume after 120 min of transient middle cerebral artery occlusion in sham, control (CON), YC-1(3-(50-hydroxymethyl-20-furyl)-1-benzylindazole, orexin-A (OXA), and orexin-A + YC-1 groups. Neurologic scores are presented as median (range); data for infarct volume are expressed as mean ± SD. Neurologic scores were evaluated at 24 h (A ) and at 72 h (B ) after reperfusion using the Garcia scoring system. (C ) Representative 2,3,5-triphenyltetrazolium chloride staining of the cerebral infarct in the rat brain at 72 h after reperfusion. (D ) Statistical analysis of the percentage of infarct volume was determined. *P < 0.05 versus  control. †P < 0.05 versus  orexin-A.

Fig. 11.  Neurologic scores and infarct volume after 120 min of transient middle cerebral artery occlusion in sham, control (CON), YC-1(3-(50-hydroxymethyl-20-furyl)-1-benzylindazole, orexin-A (OXA), and orexin-A + YC-1 groups. Neurologic scores are presented as median (range); data for infarct volume are expressed as mean ± SD. Neurologic scores were evaluated at 24 h (A ) and at 72 h (B ) after reperfusion using the Garcia scoring system. (C ) Representative 2,3,5-triphenyltetrazolium chloride staining of the cerebral infarct in the rat brain at 72 h after reperfusion. (D ) Statistical analysis of the percentage of infarct volume was determined. *P < 0.05 versus  control. †P < 0.05 versus  orexin-A.

Close modal

Representative coronal brain sections stained with 2% (w/v) 2,3,5-triphenyltetrazolium chloride from the five groups are shown in figure 11C. The mean infarction volume percentage in the orexin-A + YC-1 group is 43.2%, which was significant higher than orexin-A group (P = 0.013). There was no significant difference between control group and the orexin-A + YC-1 group in neurologic scores or infarction volume (fig. 11D).

Effect of YC-1 on HIF-1α Protein Expression

Orexin-A treatment (30 μg/kg) significantly increased the expression of HIF-1α in the hippocampus at 24 and 72 h after reperfusion when compared with the control group (P < 0.001). YC-1 treatment significantly suppressed the elevation of HIF-1α expression in the orexin-A + YC-1 group (P < 0.001 vs . orexin-A) as well as in YC-1 group (fig. 12A; P < 0.001 vs . control).

Fig. 12.  Protein expression of hypoxia-inducible factor-1α (HIF-1α) in the hippocampus (A ) and penumbral cortex (B ) in sham, control (CON), YC-1(3-(50-hydroxymethyl-20-furyl)-1-benzylindazole, orexin-A (OXA), and orexin-A + YC-1 groups were evaluated by Western blot analysis. Representative bands and corresponding β-actin bands (top ) and analysis of HIF-1α expression (bottom ) are shown for each study group. Data are presented as mean ± SD. *P < 0.05 versus  control. †P < 0.05 versus  orexin-A.

Fig. 12.  Protein expression of hypoxia-inducible factor-1α (HIF-1α) in the hippocampus (A ) and penumbral cortex (B ) in sham, control (CON), YC-1(3-(50-hydroxymethyl-20-furyl)-1-benzylindazole, orexin-A (OXA), and orexin-A + YC-1 groups were evaluated by Western blot analysis. Representative bands and corresponding β-actin bands (top ) and analysis of HIF-1α expression (bottom ) are shown for each study group. Data are presented as mean ± SD. *P < 0.05 versus  control. †P < 0.05 versus  orexin-A.

Close modal

Similar HIF-1α protein expression tendency was observed in the penumbral cortex (fig. 12B).

In the current study, we demonstrate that orexin-A treatment can induce prominent neuroprotective effects on transient cerebral ischemia in rats by improving neurologic function and decreasing infarct size. Ischemia reperfusion-induced neuronal apoptosis was also markedly attenuated by administration of orexin-A. The results of the current study also indicate that the neuroprotective effects of exogenous orexin-A treatment are mediated by HIF-1α activation and vHL inhibition.

Orexins (orexin-A and orexin B) are novel neuropeptides. Neuronal fibers of the orexin-containing neurons are widely distributed throughout the brain.1,26Previous reports have revealed that orexins are involved in the regulation of many neurologic functions such as wakefulness, appetite, and metabolism.3The physiologic function of orexins is mediated through two receptor types, the OX1R and the orexin 2 receptor.27–29OX1R and the orexin 2 receptor are G protein-coupled receptors, which transmit information into cells by activating heterotrimeric G proteins. Activation of the signaling pathways associated with distinct G proteins may contribute to the diverse physiologic roles of orexin in particular neurons. Ramanjaneya et al.  30found both extracellular receptor kinase 1/2 and p38 activation were predominantly Gq-mediated and, to a lesser extent, Gi-mediated; these two signaling pathway were phosphorylated rapidly in response to orexin-A and orexin B. The multiple roles for orexin mediated mitogen-activated protein kinase activation may help to explain the diverse biologic actions of orexins. Orexin-A increases cell surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propanoic acid receptors and potentiates α-amino-3-hydroxy-5-methyl-4-isoxazole propanoic acid receptors' mediated synaptic transmission in the striatum, providing a potential link between the activation of orexin signaling in the striatum in response to addictive substances and neural adaptations in the reward circuitry.31A recent study32also revealed that orexin-A, but not orexin B, induces a state-dependent long-term potentiation of synaptic transmission in hippocampal slices from adult mice. Long-term potentiation of synaptic transmission is blocked by pharmacologic inhibition of OX1R and plasticity-related kinases, including serine/threonine (calmodulin kinase II, protein kinase C, protein kinase A, mitogen-activated protein kinase), lipid, phosphatidylinositol 3-kinase, and receptor tyrosine kinases. Peltonen et al.  33also found orexin-A acting at OX1R-triggered oscillatory Ca2+responses through transient receptor potential channel 3, which provides a novel mechanism for sustained activation of cellular processes.

Orexins derive from a single precursor prepro-orexin. The prepro-orexin gene is located on chromosome 17, which contains genes relevant to neurodegenerative diseases.1Therefore, it is speculated that the orexin system may be involved in maintaining neuronal survival and brain integrity. A number of clinical studies have shown that sleep patterns are altered in stroke patients. For example, many patients with stroke were reported to have incidents of insomnia,34although strokes are also associated with hypersomnia.8Because the orexin system plays an important role in sleep regulation, a change in the sleep-arousal cycle after a stroke indicate that orexin signal activities may be affected by cerebral ischemia-reperfusion injury. Recently, it was reported that cerebral ischemia induces increased cortical expression of the OX1R, suggesting involvement of the orexin system in response to an ischemic insult.16Irving et al.  14reported that gene and protein expression of OX1R, but not the orexin 2 receptor, was increased in the ischemic cortex after focal ischemia in the rat. Furthermore, a decrease in orexin-A concentrations was observed in several neurodegenerative diseases and trauma in humans.35As orexin-A has a higher affinity than orexin B to bind with OX1R1and it can cross the blood-brain barrier rapidly by simple diffusion,36we have only focused on whether exogenous orexin-A is a promising neuroprotective agent against cerebral ischemia-reperfusion injury.

In this study, we have demonstrated that orexin-A treatment at the onset of reperfusion can induce neuroprotection by attenuating infarct volume and improving neurologic function in a transient rat MCAO model. These effects occurred in a dose-dependent manner at 10–30 μg/kg orexin-A. However, administration of 100 μg/kg orexin-A did not further improve the neuroprotective effects of orexin-A. This result indicates that the neuroprotective effects of orexin-A have a ceiling effect. The results of the neurologic function assessment also correlated with the reduction in infarct volume. Therefore, from the neurologic and histologic outcomes, we conclude that 30 μg/kg orexin-A treatment provides optimal neuroprotective effects. We also demonstrated these effects could exist as long as 7 days after MCAO regardless of administrative timing.

To evaluate the antiapoptotic effect of orexin-A treatment, TUNEL staining was performed in the penumbral cortex in the current study. Apoptotic cells appeared in the penumbral cortex at 2 h after ischemia reperfusion in the rat. At 24 and 72 h after reperfusion, an increased number of apoptotic neurons was observed in the control group as a result of ischemia-reperfusion injury. Administration of orexin-A at the onset of reperfusion significantly reduced the number of TUNEL-positive cells after reperfusion. These results indicate that orexin-A has an antiapoptotic function that may contribute to its neuroprotective effects.

HIF-1α is a transcription factor that has proven important for cell survival under hypoxic ischemia. HIF-1α increases the expression of HIF-1α target genes, such as vascular endothelial growth factor37and erythropoietin,38which have been shown to mediate neuroprotective effects. HIF-1α-induced angiogenesis and glycolytic metabolism result in the increased delivery of oxygen and nutrition that is critical for neuron survival under hypoxic and ischemic conditions.39This protein has been linked to homeostatic regulation of metabolism,40a function common to orexins. In addition, an in vitro  experiment15revealed that orexins can activate HIF-1α under normoxic conditions, and that this activation is partly the result of a down-regulation of vHL, an E3 ubiquitin ligase that mediates turnover of HIF-1αvia  the ubiquitin-proteasome pathway. Therefore, we wanted to know whether the neuroprotective effects induced by orexin-A are relevant to HIF-1α regulation.

As shown in the results of western blot analysis and immunohistochemistry, the expression of HIF-1α increased significantly in the penumbral cortex and hippocampus CA1 region at 2, 24, and 72 h after reperfusion in the control group when compared with the sham group. These findings are consistent with previous reports. Li et al.  41have demonstrated that HIF-1α expression increased in the hippocampus and cortex at 6 h after ischemia, peaked at 24 h, and persisted for 96 h after reperfusion in a rat model of global ischemia. Another study42reported a marked increase in HIF-1α proteins by 20 h after focal cerebral ischemia reperfusion. It is noteworthy that we found that treatment with orexin-A at the onset of reperfusion resulted in a further enhancement of HIF-1α expression, which increased at 24 and 72 h after reperfusion as compared with the control group.

To further demonstrate the involvement of the HIF-1α pathway in the neuroprotective effects of orexin-A, HIF-1α inhibitor YC-1 was used. Our results showed that YC-1 significantly suppressed increased HIF-1α expression induced by orexin-A and reversed the neuroprotective effects of orexin-A. These findings support our hypothesis that the neuroprotective effects of orexin-A may be mediated by the up-regulation of HIF-1α.

Under normoxic conditions, HIF-1α protein concentrations are low as a result of proteasomal degradation by vHL. However, HIF-1α escapes from this degradation during hypoxia, a process that is recognized as the hypoxic pathway.43,44Recently, it was revealed that HIF-1α can also be induced via  a nonhypoxic pathway by angiotensin II, tumor necrosis factor-α, and nitric oxide.45–48Kakinuma et al.  49reported that in acute myocardial ischemia, vagal nerve stimulation increased HIF-1α expression and reduced infarct size. Therefore, it is speculated that neurons possess a similar system for regulating HIF-1α through orexin-A, independent of oxygen concentration. Our current results indicate that the neuroprotective effects of orexin-A against ischemic stress can be attributed to additional HIF-1α induction.

It has been demonstrated that orexin-A can induce HIF-1α expression by regulating vHL under normoxic conditions.18To explore whether orexin-A-induced increase of HIF-1α expression is accompanied with vHL regulation, we observed protein expression of vHL at 2, 24, and 72 h after reperfusion. Western blot analysis showed that vHL protein concentrations in the penumbral cortex and hippocampus in the control group did not change significantly after ischemia reperfusion when compared with the sham group. Nevertheless, vHL protein concentrations decreased at 24 and 72 h after reperfusion in rats treated with orexin-A. These results revealed that orexin-A may reduce vHL protein expression after administration in vivo .

Our current study confirmed that increased HIF-1α concentrations, as triggered by orexin-A, have neuroprotective effects in acute transient cerebral ischemia. It is conceivable that HIF-1α induction is a powerful cellular response against hypoxic ischemia, and that additional induction of HIF-1α during ischemia via  a nonhypoxic pathway could provide further protection. We also revealed that the increase of HIF-1α by orexin-A administration was related to a reduction in the expression of vHL.

There are some limitations to the current study. First, the underlying mechanism of how orexin-A modulates vHL expression remains unclear. Further studies are needed to demonstrate whether other factors, such as HIF-1α gene transcription and prolyl-hydroxylase activity, also contribute to HIF-1α accumulation. Second, although the intraluminal suture MCAO model is the most frequently used model among experimental ischemic stroke models, it has some shortcomings. Hyperthermia occurs in most animals subjected to suture occlusion of MCAO lasting 2 h or more. This phenomenon appears to be associated with hypothalamic injury. The stroke condition induced by this model is different from the clinical stroke situation in some aspects. Withdrawal of the intraluminal thread induces instantaneous reperfusion whereas spontaneous or thrombolysis-induced recanalisation results in slowly progressing recirculation. As postischemic recovery is greatly influenced by the dynamics of reperfusion, the outcome and pharmacologic responsiveness of transient filament occlusion is distinct from most clinical situations of reversible ischemia.

In summary, we have demonstrated for the first time that exogenous orexin-A can induce neuroprotective effects against cerebral ischemia-reperfusion injury. Furthermore, we have shown that the neuroprotection of orexin-A may be mediated through the HIF-1α pathway. Therefore, it is imperative to determine the mechanism of neuroprotection of orexin-A, which may be a novel neuroprotectant for future use. Further experiments using orexin-deficient animals will be extremely useful.

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