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Hypoxic preconditioning attenuates global cerebral ischemic injury following asphyxial cardiac arrest through regulation of delta opioid receptor system
Neuroscience 202 (2012) 352–362
HYPOXIC PRECONDITIONING ATTENUATES GLOBAL CEREBRAL ISCHEMIC INJURY FOLLOWING ASPHYXIAL CARDIAC ARREST THROUGH REGULATION OF DELTA OPIOID RECEPTOR SYSTEM C.-J. GAO,a1 L. NIU,b1 P.-C. REN,c1 W. WANG,b C. ZHU,b Y.-Q. LI,b* W. CHAIa* AND X.-D. SUNa*
Key words: hypoxic preconditioning, neuroprotection, cardiac arrest, delta opioid receptor, hypoxia-inducible factor-1␣.
a
Department of Anesthesiology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province 710038, China
Ischemic brain injury, resulting from global cerebral ischemia after cardiac arrest (CA) and successful resuscitation, is one of the most common and important causes of disability and death (Böttiger et al., 1999; Vogel et al., 2003; Jia et al., 2008; Dirnagl et al., 2009). Poor neurological outcomes after CA and resuscitation bring heavy burdens on family and society. Although there are large numbers of neuroprotective approaches that have been developed to mitigate ischemic cerebral injury and improve neurological outcomes following CA in laboratory experiments, no specific and reliable therapy is available up to now (Safar, 2000; Popp et al., 2007; Teschendorf et al., 2008). Substantial data has demonstrated that hypoxic preconditioning (HPC) could induce tolerance to cerebral ischemia/reperfusion injury, even in global cerebral ischemia following CA and resuscitation (Geocadin et al., 2005; Li et al., 2007). However, the underlying mechanisms are not fully understood and more evidences are needed for HPCmediated neuroprotection, especially in global cerebral ischemia following CA and resuscitation. Large amounts of data have indicated that delta opioid receptor (DOR) plays a crucial role in maintaining neuronal survival, and DOR activation is indeed neuroprotective against hypoxic or ischemic stress, either in in vitro neurons or in vivo models of brain ischemia (Zhang et al., 2002; Ma et al., 2005; Narita et al., 2006; Iwata et al., 2007; Su et al., 2007; Yang et al., 2009; Zhu et al., 2009). Previous studies have also showed that HPC upregulates DOR, and induces long-term protection either in cortical neurons against severe hypoxia, or in retina against elevated intraocular pressure (IOP) (Ma et al., 2005; Zhang et al., 2006; Peng et al., 2009). We therefore examined whether DOR is involved in the mediation of the neuroprotective effect induced by HPC in asphyxial CA rat model. In addition, it is well known that HPCinduced ischemia tolerance is profoundly dependent on hypoxia-inducible factor-1␣ (HIF-1␣) (Neubauer, 2001; Geocadin et al., 2005; Peng et al., 2009; Taie et al., 2009), then we further investigated the relationship between HIF-1␣ and DOR in the HPC-treated brain.
b
Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Center, Fourth Military Medical University, Xi’an, Shaanxi Province 710032, China
c
Department of Orthopaedics, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province 710038, China
Abstract—This study was designed to investigate whether delta opioid receptor (DOR) is involved in the neuroprotective effect induced by hypoxic preconditioning (HPC) in the asphyxial cardiac arrest (CA) rat model. Twenty-four hours after the end of 7-day HPC, the rats were subjected to 8-min asphyxiation and resuscitated with a standardized method. In the asphyxial CA rat model, HPC improved the neurological deficit score (NDS), inhibited neuronal apoptosis, and increased the number of viable hippocampal CA1 neurons at 24 h, 72 h, or 7 days after restoration of spontaneous circulation (ROSC); however, the above-mentioned neuroprotection of HPC was attenuated by naltrindole (a selective DOR antagonist). The expression of hypoxia-inducible factor-1␣ (HIF-1␣) and DOR, and the content of leucine enkephalin (L-ENK) in the brain were also investigated after the end of 7-day HPC. HPC upregulated the neuronal expression of HIF-1␣ and DOR, and synchronously elevated the content of L-ENK in the rat brain. HIF-1␣ siRNA was used to further elucidate the relationship between HIF-1␣ and DOR in the HPC-treated brain. Knockdown of HIF-1␣ by siRNA markedly abrogated the HPC induced upregulation of HIF-1␣ and DOR. The present study demonstrates that the expression of DOR in the rat brain is upregulated by HIF-1␣ following exposure to 7-day HPC, at the same time, HPC also increases the production of endogenous DOR ligand L-ENK in the brain. DOR activation after HPC results in prolonged neuroprotection against subsequent global cerebral ischemic injury, suggesting a new mechanism of HPC-induced neuroprotection on global cerebral ischemia following CA and resuscitation. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. 1
Contributed equally to this work. *Corresponding author. Tel: ⫹86-02984774501 or ⫹86-02984777439 or ⫹86-02984777539. E-mail address: [email protected] (Y.-Q. Li) or tdmzka@ fmmu.edu.cn (W. Chai) or [email protected] (X.-D. Sun). Abbreviations: ACSF, artificial cerebrospinal fluid; CA, cardiac arrest; CPR, cardiopulmonary resuscitation; Ct, threshold cycle; DADLE, [DAla2, D-Leu5] enkephalin; DOR, delta opioid receptor; HIF-1␣, hypoxiainducible factor-1␣; HPC, hypoxic preconditioning; L-ENK, leucine enkephalin; NDS, neurological deficit score; NTI, naltrindole; PB, phosphate buffer; PBS, phosphate buffered saline; ROSC, restoration of spontaneous circulation; RT-PCR, reverse transcription polymerase chain reaction; TBS, Tris-buffered saline; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling.
EXPERIMENTAL PROCEDURES Animal preparation This study was approved by the Animal Care and Use Committee of the Fourth Military Medical University (Xi’an, China), and followed the National Guidelines for Animal Experimentation. Male
0306-4522/12 $36.00 © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.11.060
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C.-J. Gao et al. / Neuroscience 202 (2012) 352–362 Sprague–Dawley rats weighing between 300 and 350 g were obtained from the experimental animal center of our university. Animals were individually housed and maintained on a 12 h light and dark cycle with free access to water and standard rat chow. Every effort was made to minimize animal suffering and reduce the number of animals used.
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ACSF⫹CA group and HPC⫹NTI⫹CA group received i.c.v. infusion of 10 l ACSF containing 0 or 50 nmol naltrindole 30 min before the induction of CA. The doses of naltrindole were determined according to our preliminary experiments. Survival rate after CA and CPR were calculated.
Neurological evaluation Induction of hypoxic preconditioning The day before the start of HPC, rats were acclimated to the custom-made Plexiglas chambers (one rat per chamber, 12⫻5⫻5 in.3, gas flow through chamber⫽4 L/min) under normoxic conditions. Within the chambers, O2 levels were continuously monitored with an S-3A Oxygen Analyzer (Applied Electrochemistry Inc., Sunnyvale, CA, USA). To induce HPC, rats were placed daily into the Plexiglas chambers in which oxygen content was reduced to 10% by continuously flushing with nitrogen from 0800 h– 0000 h daily for 7 days. Control animals were kept in the chambers under normoxic conditions. The animals were allowed free access to food and water during HPC.
Delta opioid receptor inhibition After anesthetizing by i.p. injection of 3% pentobarbital sodium (40 mg/kg), the rats were placed in a stereotaxic frame (SN-1C, Narishige Instruments, Tokyo, Japan) with a horizontal skull position. Then, 10 l artificial cerebrospinal fluid (ACSF) containing 50 nmol naltrindole (NTI, a selective DOR antagonist; Sigma, St Louis, MO, USA) was administered into the left cerebroventricle with a Hamilton microsyringe (80630, Hamilton Co., Reno, NV, USA), using the following coordinates: 0.8 mm posterior to bregma, 1.4 mm lateral to the midline on the left side, and 4.0 mm ventral to the skull surface. All coordinates were derived from according to the coordinates of the Paxinos and Watson atlas (Paxinos and Watson, 2004).
Cardiac arrest and cardiopulmonary resuscitation The procedure of CA and cardiopulmonary resuscitation (CPR) was performed as our previous study (Gao et al., 2010). Briefly, under anesthesia with 3% pentobarbital sodium (i.p. 40 mg/kg), each rat was intubated tracheally with a 14-gauge cannula and ventilated by a volume controlled small animal ventilator (DHX150, Medical Instrument Factory of Chengdu, China) with room air to maintain normocapnia. The arterial blood pressure (ABP) and electrocardiogram (ECG) were monitored. Asphyxia was induced by stopping mechanical ventilation and clamping the tracheal tubes at the end of expiration. After 8-min asphyxiation, CPR was initiated by administering a bolus injection of epinephrine (0.02 mg/kg, i.v.) and 5% sodium bicarbonate (1 mmol/kg, i.v.) followed by mechanical ventilation and thoracic compressions. Restoration of spontaneous circulation (ROSC) was defined as the return of supraventricular rhythm with an increase of mean artery pressure (MAP) beyond values of 60 mm Hg. Ventilation was consistently maintained until spontaneous breath started. Failure to restore spontaneous circulation within 10 min resulted in discontinuation of resuscitation efforts.
Experiment I To evaluate the effect of DOR inhibition on neuroprotection induced by HPC, rats were randomly assigned to CA, HPC⫹CA, NTI⫹CA, HPC⫹ACSF⫹CA, and HPC⫹NTI⫹CA groups (20 rats for each group). Animals in the CA group were only subjected to 8-min asphyxiation and CPR. Animals in NTI⫹CA group received ICV infusion of 50 nmol naltrindole 30 min before the induction of CA. All the rats in other three groups received 7-day HPC. At 24 h after the end of HPC, animals in HPC⫹CA group were subjected to 8-min asphyxiation and CPR, whereas animals in HPC⫹
To evaluate the neurological outcome in the successfully resuscitated animals (n⫽7 for each group), the neurological deficit score (NDS) was determined at 24 h, 72 h, and 7 days after ROSC according to previous studies (Geocadin et al., 2000; Jia et al., 2008). In brief, scoring includes seven parameters: general behavior, brain stem function, motor and sensory function assessment, motor behavior (including gait coordination and balance on beam), other behavior reflexes, and seizures. The NDS and its components are presented in Table 1. An NDS of 80 reflected Table 1. Neurological deficit Score (NDS) for rats (A) General behavioral Consciousness Arousal
Respiration
Total score⫽19 Normal [10], stuporous [5], comatose [0] Eyes open spontaneously [3], eyes open to pain [1], no eye opening [0] Normal [6], abnormal [0], absent [0]
(B) Brain-stem function Olfaction Vision Pupillary reflex Corneal reflex Startle reflex Whisker stimulation Swallowing
Total score⫽21 Present [3], absent Present [3], absent Present [3], absent Present [3], absent Present [3], absent Present [3], absent Present [3], absent
(C) Motor assessment Strength (left and right side tested and scored separately)
Total score⫽6 Normal [3], stiff/weak [1], no movement/paralyzed [0]
(D) Sensory assessment Pain (left and right side tested and scored separately)
Total score⫽6 Brisk withdrawal with pain [3], weak or abnormal response [1], no withdrawal [0]
(E) Motor behavior Gait coordination Balance on beam
Total score⫽6 Normal [3], abnormal [1], absent [0] Normal [3], abnormal [1], absent [0]
(F) Behavior Righting reflex Negative geotaxis Visual placing Turning alley
Total score⫽12 Normal [3], abnormal Normal [3], abnormal Normal [3], abnormal Normal [3], abnormal
(G) Seizures Convulsive or nonconvulsive
Total score⫽10 No seizure [10], focal seizure [5], general seizure [0]
[0] [0] [0] [0] [0] [0] [0]
[1], [1], [1], [1],
absent absent absent absent
[0] [0] [0] [0]
Balance beam testing is normal if the rat can cross a 2 cm wide by 1 m long beam suspended 0.5 m above the floor. Abnormal is scored if the rat attempts and does not continue or stays momentarily and falls. Absent is scored when the rat falls off immediately upon placement on the beam. Other behavior reflex subscores evaluated the following: (1) righting reflex (animal placed on its back is able to correct to upright position); (2) turning alley (the animal is made to walk and turn back at the end of a 15 cm⫻0.5 m alley); (3) visual placing (the animal is lifted and is able to visually orient itself to objects and depth); (4) negative geotaxis (animal placed on its back on a plane angled at 45° corrects itself and moves up the incline).
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normal brain function, whereas an NDS of 0 represented brain death. The evaluation was always carried out by the same investigator who was blinded to the experimental groups.
Histological examination At the 7th day after ROSC, the rats (n⫽7 for each group) were deeply anesthetized and transcardially perfused with 100 ml 0.9% saline followed by 500 ml 0.1 M phosphate buffer (PB, pH 7.4) that contained 4% paraformaldehyde and 2% picric acid. Brains were removed and post-fixed in the same fixative for 2– 4 h and then cryoprotected for 24 h at 4 °C in 0.1 M PB that contained 30% sucrose. Serial coronal sections (10 m) from the area located at 3.5 mm posterior to bregma were subsequently obtained using a Leica cryostat (Leica CM1800, Heidelberg, Germany) and stained with Cresyl Violet. The viable neurons in the hippocampal CA1 region were observed with an Olympus CH30 microscope (Olympus, Corporation; magnification 400-fold) attached to an Olympus DP70 digital microscope camera (Olympus America Inc., Melville, NY, USA). The viable neurons were considered to be those with visible nucleus and intact cytoplasm with discernable and rich Nissl staining. Neurons that had shrunken cell bodies with surrounding empty spaces were excluded. The viable neurons in the hippocampal CA1 region were observed with an Olympus CH30 microscope (Olympus, Corporation).
TUNEL staining Twenty-four hours after ROSC, samples from five groups (n⫽7 for each group) in Experiment I were used. The coronal sections were obtained as described in the histological examination experiment. Neuronal apoptosis in the hippocampus CA1 region was assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining using an in situ cell death detection kit (TMR Red, Roche, Mannheim, Germany) according to the manufacturer’s recommendations. TUNEL-positive neurons in the CA1 region were observed using the Olympus BX-60 fluorescence microscope (Olympus Corporation). As described by Yamashiro et al. (2007), the number of viable neurons and TUNEL-positive neurons in CA1 region were counted in the predefined area (0.25 mm2) at a magnification of 400-fold by an investigator blinded to the experimental groups, using AxioVision software (Zeiss, Jena, Germany).
Experiment II To determine the regulatory effect of HPC on the delta opioid system and HIF-1␣ before cardiac arrest, rats were randomly divided into two groups: Control group and HPC group. At 24 h after the end of HPC, three rats from each group were sacrificed for immunofluorescence staining. At 1 day, 3 days, or 7 days after the end of HPC, the brains were harvested for real-time reverse transcription polymerase chain reaction (RT-PCR), Western blot, or measurement of leucine enkephalin (n⫽5 for each time point).
Immunofluorescence staining The coronal sections were obtained as described in Experiment I. The 10 m thick coronal sections were incubated with primary antibodies: goat anti-DOR (1:100; Santa Cruz Biotechnology, CA, USA) and mouse anti-HIF-1␣ (1:100; Santa Cruz) for 48 h at 4 °C. The sections were washed three times in 0.01 M phosphate buffered saline (PBS; 10 min each) and then incubated for 6 h at room temperature with the secondary antibodies: FITC (fluorescein isothiocyanate)-labeled rabbit anti-goat IgG for DOR (1:200; Vector, Burlingame, CA, USA) and Cy3 (cyanin 3)-labeled donkey anti-mouse IgG for HIF-1␣ (1:200; Vector). After staining, the sections were then rinsed in PBS, air dried, coverslipped with a mixture of 50% glycerin and 2.5% triethylene diamine in PBS, and
observed with a confocal laser scanning microscope (Olympus FV1000, Tokyo, Japan).
Real-time RT-PCR Total RNA was extracted from the cortex and hippocampus using a Trizol kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was treated with RNase-free DNase I (Takara Biotechnology, Dalian, China) and reverse transcribed (RT) into cDNA with PrimeScript™ RT reagent Kit (Takara). The cDNA was then used as a template for real-time PCR with the primers for HIF-1␣: 5=-CCAGATTCAAGATCAGCCAGCA-3= (forward) and 5=-GCTGTCCACATCAAAGCAGTACTCA-3= (reverse), DOR: 5=-AACGTGCTCGTCATGTTTGG-3= (forward) and 5=-CAGGTACTTGGCGCTCTGGAA-3= (reverse), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase): 5=-GGCACAGTCAAGGCTGAGAATG-3= (forward) and 5=-ATGGTGGTGAAGACGCCAGTA-3= (reverse). Real-time RT-PCR reactions were performed in MiniOpticon Real-Time PCR Detection System (Bio-Rad Laboratories, CA, USA) using the SYBR Green RealTime PCR Master Mix (Toyobo Co. Ltd., Japan). PCR products were electrophoresed in 2% agarose gel (Invitrogen) to confirm that PCR yielded a single product of the expected size. The expected sizes were 100 bp (HIF-1␣), 133 bp (DOR), and 143 bp (GAPDH). The threshold cycle (Ct) was defined for each amplification plot. Each sample was run in triplicate and mean Ct values were used for further calculations. The results were analyzed by the 2⫺⌬⌬Ct method (Livak and Schmittgen, 2001). To ensure validity of our calculations, we confirmed that primer sets used in this study have the same efficiencies as ascertained by varying template concentrations. In each case, the log of the template concentration when plotted against ⌬Ct yielded values of less than 0.1 for the slope. The fold changes of mRNA levels in all samples were expressed relative to the calibrator (100%). The value of the control sample served as calibrator.
Western blot Western blot analysis was performed as described previously (Persson et al., 2005). Tissues from the cortex and hippocampus were homogenized for 20 min in lysis buffer containing Complete™ protease inhibitor cocktail (Santa Cruz Biotechnology) on ice. The samples were centrifuged for 25 min at 15,000 r/min at 4 °C. The supernatant containing total protein was collected, and the protein concentration was determined by Bradford method. The electrophoresis samples were heated at 100 °C for 5 min and loaded onto 10% SDS-polyacrylamide gel with standard Laemmli solutions (Bio-Rad Laboratories, CA, USA). The proteins were electroblotted onto polyvinylidene difluoride membranes (PVDF, Immobilon-P, Millipore, Billerica, MA, USA). The membranes were placed in a blocking solution, which contained Tris-buffered saline with 0.02% Tween (TBS-T) and 5% non-fat dry milk, for 1 h, and incubated overnight under gentle agitation with primary antibodies: goat anti-DOR (1:100; Santa Cruz), mouse anti-HIF-1␣ (1: 100; Santa Cruz), and rabbit anti--actin (1:5000; Sigma, St Louis, MO, USA). Bound primary antibodies were detected with the anti-goat, anti-mouse, or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). All reactions were detected by the enhanced chemiluminescence (ECL) detection method (Amersham). The densities of protein blots were analyzed by using Labworks Software (Ultra-Violet Products Ltd., Cambridge, UK) and normalized to -actin levels.
Measurement of leucine enkephalin The brain content of leucine enkephalin (L-ENK) was determined using a commercial kit (Phoenix Pharmaceuticals, Belmont, CA, USA) as described previously after peptide extraction (Ma et al., 2005). The values of L-ENK are expressed as ng/mg of protein.
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Experiment III To further elucidate the relationship between HIF-1␣ and DOR in the HPC-treated brain, rats were randomly divided into HIF-1␣ siRNA group and control siRNA group (n⫽5 for each group). At 24 h after the end of 7-day HPC, we performed in vivo HIF-1␣ siRNA transfer according to the method described previously (Chen et al., 2009). Then, 10 l HIF-1␣ siRNA (sc-45919, Santa Cruz) or control siRNA-A (sc-37007, Santa Cruz) was diluted with the same volume of transfection reagent (sc-29528, Santa Cruz) and mixed gently. After incubation for 45 min at room temperature, the mixture was injected intraparenchymally using a Hamilton microsyringe under the guidance of a stereotaxic frame (SN-1C) and the infusion was delivered over 60 min. The protein expressions of HIF-1␣ and DOR in the parietal cortex around the injection point were evaluated 24 h post-transfection using Western blot and immunofluorescence staining as described earlier in the text.
Statistical analysis The software, SPSS 13.0 for Windows (SPSS Inc., Chicago, USA), was used to conduct statistical analyses. All values, except for survival rate and neurological deficit score, are presented as mean⫾SD and were analyzed by one-way analysis of variance, and between-group differences were detected with post hoc Student–Newman–Keuls test. Survival rate comparison was done using the Fisher’s exact test. The neurological deficit scores were expressed as median (range) and analyzed with Kruskal–Wallis test followed by the Mann–Whitney U-test with Bonferroni correction. Values of P⬍0.05 were considered statistically significant.
RESULTS Survival rate The survival rate in the CA group was 75.0% (15/20 rats); in the HPC⫹CA group 90.0% (18/20 rats); in the NTI⫹CA group 70.0% (14/20 rats); in the HPC⫹ACSF⫹CA group 85.0% (17/20 rats); in the HPC⫹NTI⫹CA group 75.0% (15/20 rats). Statistical analysis revealed that there were no significant differences of the survival rate among all the groups using the Fisher exact test. Data collected from rats that did not survive were not included in the following analysis. Physiological variables and characteristics Before induction of CA, there were no differences in body weight, temperature, arterial blood gas, and hemodynamic variables among all the groups (data not shown). Parameters related to the resuscitation procedures, such as MAP, heart rate, and arterial blood gas data during CPR and the early ROSC phase did not differ in the five groups (data not shown). HPC-induced improvement of neurological outcome is reversed by naltrindole The NDS was determined at 24 h, 72 h, and 7 days after ROSC and the results are shown in Fig. 1. There were significant differences among the five groups with regard to the neurological status at all the three time points after ROSC (P⬍0.05). Compared with the CA group, the ani-
Fig. 1. Neurological deficit score (NDS) was presented as median and 25th–75th percentile (n⫽8). Significant differences were observed among the five groups with regard to the neurological status at all the three time points after ROSC. HPC significantly improved the neurological deficit scores, whereas the DOR antagonist naltrindole reversed the beneficial effect of HPC. * P⬍0.01 versus CA group; # P⬍0.01 versus HPC⫹CA group.
mals in the HPC⫹CA group had persistently better functional recovery in NDS at all time points with statistical significance (P⬍0.01). The DOR antagonist naltrindole had no effect on NDS when administered alone but reversed the beneficial effect of HPC given 30 min before the onset of CA (P⬍0.01, HPC⫹NTI⫹CA versus HPC⫹CA). The NDS of the HPC⫹ACSF⫹CA group was similar to that of the HPC⫹CA group. There were no statistical differences in NDS among the CA, NTI⫹CA, and HPC⫹ NTI⫹CA groups. HPC-induced improvement of morphology changes is abolished by naltrindole Compared with the sham animal, histological evaluation of the hippocampal CA1 region revealed various degrees of neuronal damage in all the surviving animals following CA and CPR (Fig. 2A). However, the number of viable neurons in the hippocampal CA1 region was significantly increased in the HPC⫹CA group in comparison with the CA group as revealed by Nissl staining (P⬍0.01, Fig. 2B); there was no difference between the HPC⫹ACSF⫹CA and HPC⫹CA groups. The number of viable neurons in the NTI⫹CA group was similar to that in the CA group. Naltrindole reduced the number of viable neurons in the HPC⫹ NTI⫹CA group compared with the HPC⫹CA group (P⬍0.05, Fig. 2B). HPC-induced reduction of neuronal apoptosis is attenuated by naltrindole No positive TUNEL staining (red) neuron was detected in the brain sections of sham animals. However, a large number of TUNEL-positive neurons in the hippocampal CA1 region of rat brain were seen in the CA, NTI⫹CA, and HPC⫹NTI⫹CA groups, whereas in contrast, only a small amount of TUNEL-positive neurons in the HPC⫹ACSF⫹
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Fig. 2. (A) Representative photomicrographs (Nissl staining; magnification 400-fold) showing the viable neurons in hippocampal CA1 region at the 7th day after ROSC. (B) The number of viable neurons in the hippocampal CA1 region detected through Nissl staining. HPC significantly increased the number of viable neurons, whereas the DOR antagonist naltrindole abolished the protective effect of HPC. * P⬍0.01 versus CA group; # P⬍0.05 versus HPC⫹CA group. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
CA and HPC⫹CA groups were observed (Fig. 3A). The quantitative analysis of the number of TUNEL-positive neurons in the hippocampal CA1 region showed that HPC significantly reduced the number of TUNEL-positive neurons compared with the CA and NTI⫹CA groups (P⬍0.01). Naltrindole reversed the beneficial effect of HPC given 30 min before the onset of CA (P⬍0.05, HPC⫹NTI⫹CA versus HPC⫹CA, Fig. 3B). HPC upregulates the expression of HIF-1␣ and DOR in the brain The changes in the mRNA and protein levels of HIF-1␣ were paralleled in groups (Figs. 4A and 5A). The expression of HIF-1␣ mRNA and protein in both the cortex and hippocampus significantly increased at 1 day and 3 days after the end of 7-day HPC as compared with those in the control groups (Figs. 4B and 5B, P⬍0.05), but returned to normal level at 7 days after the end of 7-day HPC. HPC also produced evident upregulation of DOR expression in the brain as revealed by the presence of both mRNA (Fig. 4A) and protein (Fig. 5A). The expression of DOR mRNA
and protein in the hippocampus and cortex significantly increased at 1 day and 3 days after the end of 7-day HPC as compared with those in the control group (Figs. 4C and 5C, P⬍0.05), but returned to normal level at 7 days after the end of 7-day HPC. The expression of HIF-1␣ and DOR was also examined by immunofluorescence staining. As shown in Fig. 6, in the control group, little expression of HIF-1␣ was observed in the hippocampal CA1, CA3, and cortex of rat brain. However, the expression of HIF-1␣ was significantly increased after 7-day HPC induction. Compared with the control animal, DOR was mostly upregulated after 7-day HPC induction in hippocampal CA1, CA3, and cortex of rat brain. HPC increases the content of L-ENK in brain tissue Fig. 7 showed that endogenous opioid L-ENK was present in the control brain tissue. The content of L-ENK was significantly increased in the HPC-treated rat brains at 1 day and 3 days after the end of 7-day HPC; however, its
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Fig. 3. Neuronal apoptosis in hippocampal CA1 region at 24 h after ROSC. (A) Representative photomicrographs of TUNEL staining (magnification 400-fold). (B) Quantitative analysis of the number of TUNEL-positive neurons. HPC significantly decreased the number of TUNEL-positive neurons, whereas the DOR antagonist naltrindole attenuated the reduction in the number of TUNEL-positive neurons. * P⬍0.01 versus CA group; # P⬍0.05 versus HPC⫹CA group. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
level was slightly but insignificantly decreased at 7 days after the end of 7-day HPC. Knockdown of HIF-1␣ by siRNA decreases the expression of HIF-1␣ and DOR Twenty-four hours after intraparenchymal administration of HIF-1␣ siRNA or control siRNA-A, HIF-1␣ and DOR protein in rat brain were analyzed by Western blot. The protein expression of HIF-1␣ in the parietal cortex around the injection point was significantly reduced in 7-day HPC rats that were treated with HIF-1␣ siRNA when compared with the control siRNA-A. Interestingly, the expression of DOR was also decreased by HIF-1␣ siRNA (Fig. 8, P⬍0.05). The expression of HIF-1␣ and DOR was also examined by immunofluorescence staining, and the results were consistent with Western blot experiments (Fig. 9).
DISCUSSION Recently, DOR has been reported to participate in the mechanisms of endogenous neuroprotective effects of
HPC in cultured cortical neurons as well as in rat retina in vivo (Ma et al., 2005; Zhang et al., 2006; Peng et al., 2009). However, little is known about the involvement of the DOR in HPC-induced global cerebral ischemic tolerance. Asphyxial CA is one of the most important causes of global cerebral ischemia. Therefore, we used a rat model of asphyxial CA to observe neuroprotective effects of HPC and investigate the role of DOR in HPC-induced neuroprotection. The HPC protocol (10% O2, 4 h/day for 7 days) used in this study induced that kind of delayed tolerance and exerted potent neuroprotection manifested as inhibition of neuronal apoptosis and alleviation in hippocampus CA1 neuron injury as well as amelioration of neurological outcome in our asphyxial CA model. Using this asphyxial CA rat model, we demonstrated that neuroprotective effects of HPC were attenuated by a highly selective DOR antagonist, naltrindole. The results indicate that DOR plays important roles in HPC-induced neuroprotection on global cerebral ischemic injury following CA and CPR.
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lated by 5% oxygen as shown in Ma’s work (Ma et al., 2005). This conception is also supported by previous in vivo studies. Peng and colleagues have also shown that 4 weeks of mild hypoxic upregulates DOR expression in rat retinas (Peng et al., 2009). In the present study, we found that the upregulation of DOR mRNA and protein in the brain was synchronous after 7-day HPC. Both of DOR mRNA and protein expression increased at 1 day after the end of HPC and maintained at least for 3 days, then returned to normal level at 7 days after the end of HPC. Another main hypothesis tested in this study is that the content of endogenous DOR ligand in the brain would be modulated by 7-day HPC. It is interesting to note that HPC produced an elevation of the content of L-ENK in the brain tissue. Not only that, this elevated L-ENK could be maintained for at least 3 days after 7-day HPC, which was consistent with the upregulation of DOR mRNA and protein. As is well known, an
Fig. 4. The mRNA expression of HIF-1␣ and DOR was analyzed with real-time RT-polymerase chain reaction. The PCR products were demonstrated by Panel (A). Panel (B, C) showed HPC significantly upregulated the mRNA expression of HIF-1␣ and DOR in both hippocampus and cortex at 1 d and 3 d after the end of 7-d HPC. * P⬍0.05 versus Control group.
Preconditioning research is an attractive experimental strategy to identify endogenous protective mechanisms. The rapid tolerance of HPC is independent of protein synthesis and mediated by posttranslational modification, whereas the delayed effects of HPC appears to require synthesis of new RNA and protein (Barone et al., 1998; Sharp et al., 2004). An important finding of the present work was that HPC induced upregulation of the protein and mRNA expression of DOR in the hippocampus and cortex. DOR is an oxygen-sensitive protein, and DOR expression varies greatly, depending on the duration and extent of hypoxia (Ma et al., 2005; Zhang et al., 2006). Besides downregulation after prolonged and severe hypoxia, DOR expression in the cultured cortical neurons was upregu-
Fig. 5. The protein expression of HIF-1␣ and DOR was evaluated with Western blot. Representative immunoblots of HIF-1␣ and DOR were demonstrated by Panel (A). Panel (B, C) showed HPC significantly upregulated the protein expression of HIF-1␣ and DOR in both hippocampus and cortex at 1 d and 3 d after the end of 7-d HPC. * P⬍0.05 versus Control group.
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Fig. 6. Representative immunofluorescence staining for HIF-1␣ and DOR in hippocampal CA1, CA3, and cortex of rat brain at 24 h after 7-d HPC. Both HIF-1␣ and DOR expression were mostly upregulated in the HPC group compared with the Control group. Scale bar⫽20 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
increase in receptor agonists may increase receptor-mediated intracellular activity. The number of receptors may
be another major rate-limiting factor in terms of the increase in receptor-mediated activity. Thus, we can con-
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Fig. 7. Changes of leucine enkephalin in the hippocampus and cortex. HPC significantly increased the content of leucine enkephalin in the brain at 1 d and 3 d after the end of 7-d HPC. * P⬍0.05 versus Control group.
clude from the present study that HPC increases the expression of DOR and the production of the endogenous DOR ligand L-ENK, and the ligand-receptor reaction elicits protective effects against global cerebral ischemic injury following CA and CPR. Since DOR belongs to the G protein-coupled receptor family, G protein activity along with its downstream signaling pathway may be involved in the initial steps of HPC-induced neuroprotection. It has been deduced that the DOR-G protein-PKC-pERK-Bcl 2 pathway is one of the most important neuroprotective mechanisms in cortical neurons and can be activated by HPC to counteract stress-induced death signals (Ma et al., 2005). Recently, Peng and colleagues demonstrated that neuroprotective effect of HPC against IOP elevation involves the restoration of redox imbalance and the inhibition of pro-apoptotic caspase 3 after DOR-ERK activation (Peng et al., 2009). This notion provided novel insights into the mechanism of DOR-mediated HPC neuroprotection. However, the pathophysiologic responses to global cerebral
ischemia induced by asphyxial CA are inherently different from all the above situations. We believe that further studies are needed to elucidate the intracellular mechanisms underlying DOR-mediated HPC neuroprotection in global cerebral ischemia following CA. HIF-1␣ is an important transcription protein that regulates gene expression in the brain and other tissues in response to decreases in oxygen availability. Large amounts of data have demonstrated that HIF-1␣ is essential for the induction of tolerance to ischemia by HPC. It is well known that HPC induces the expression of the protein HIF-1␣ and its downstream genes such as vascular endothelial growth factor (VEGF), erythropoietin (EPO), inducible nitric oxide synthase (iNOS), and so on. However, several studies (Ma et al., 2005; Zhang et al., 2006; Peng et al., 2009) and our results indicated that DOR protein was involved in the neuroprotection of HPC. We also observed that HPC synchronously upregulated the expression of HIF-1␣ and DOR in the brain. So we further investigated the relationship between HIF-1␣ and DOR in the HPC-treated brain. An important finding was that HIF-1␣ siRNA markedly abrogated the HPC-induced upregulation of HIF-1␣ and DOR. The previously mentioned results strongly indicated that there was a close relationship between HIF-1␣ and DOR in the HPC-treated brain, and DOR may be another downstream gene of HIF-1␣. Peng et al. have also observed the similar relationship between HIF-1␣ and DOR in the HPC-treated retina (Peng et al., 2009). However, further studies are needed to reveal the precise interaction between HIF-1␣ and DOR.
CONCLUSIONS In conclusion, the expression of DOR in the rat brain is upregulated by HIF-1␣ following exposure to 7-day HPC, at the same time, HPC also increases the production of endogenous DOR ligand L-ENK in the brain. DOR activation after HPC results in prolonged neuroprotection against subsequent global cerebral ischemic injury, suggesting a
Fig. 8. Knockdown of HIF-1␣ by siRNA decreases the expression of HIF-1␣ and DOR in the parietal cortex around the injection point. Representative immunoblots of HIF-1␣ and DOR were demonstrated by Panel (A). Panel (B) showed HIF-1␣ siRNA markedly abrogated the HPC-induced upregulation of HIF-1␣ and DOR proteins. * P⬍0.05 versus Control siRNA group.
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Fig. 9. Representative immunofluorescence staining for HIF-1␣ and DOR in the parietal cortex around the injection point at 24 h post-transfection. Both HIF-1␣ and DOR expression were mostly downregulated in the HIF-1␣ siRNA group compared with the Control siRNA group. Scale bar⫽20 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
new mechanism of HPC-induced neuroprotection on global cerebral ischemia following CA and CPR. Acknowledgments—This work was supported by the National Natural Science Foundation of China (Grant. 81071527) and the Chinese Postdoctoral Science Foundation (Grant. 201003743).
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(Accepted 28 November 2011) (Available online 21 December 2011)
HYPOXIC PRECONDITIONING ATTENUATES GLOBAL CEREBRAL ISCHEMIC INJURY FOLLOWING ASPHYXIAL CARDIAC ARREST THROUGH REGULATION OF DELTA OPIOID RECEPTOR SYSTEM C.-J. GAO,a1 L. NIU,b1 P.-C. REN,c1 W. WANG,b C. ZHU,b Y.-Q. LI,b* W. CHAIa* AND X.-D. SUNa*
Key words: hypoxic preconditioning, neuroprotection, cardiac arrest, delta opioid receptor, hypoxia-inducible factor-1␣.
a
Department of Anesthesiology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province 710038, China
Ischemic brain injury, resulting from global cerebral ischemia after cardiac arrest (CA) and successful resuscitation, is one of the most common and important causes of disability and death (Böttiger et al., 1999; Vogel et al., 2003; Jia et al., 2008; Dirnagl et al., 2009). Poor neurological outcomes after CA and resuscitation bring heavy burdens on family and society. Although there are large numbers of neuroprotective approaches that have been developed to mitigate ischemic cerebral injury and improve neurological outcomes following CA in laboratory experiments, no specific and reliable therapy is available up to now (Safar, 2000; Popp et al., 2007; Teschendorf et al., 2008). Substantial data has demonstrated that hypoxic preconditioning (HPC) could induce tolerance to cerebral ischemia/reperfusion injury, even in global cerebral ischemia following CA and resuscitation (Geocadin et al., 2005; Li et al., 2007). However, the underlying mechanisms are not fully understood and more evidences are needed for HPCmediated neuroprotection, especially in global cerebral ischemia following CA and resuscitation. Large amounts of data have indicated that delta opioid receptor (DOR) plays a crucial role in maintaining neuronal survival, and DOR activation is indeed neuroprotective against hypoxic or ischemic stress, either in in vitro neurons or in vivo models of brain ischemia (Zhang et al., 2002; Ma et al., 2005; Narita et al., 2006; Iwata et al., 2007; Su et al., 2007; Yang et al., 2009; Zhu et al., 2009). Previous studies have also showed that HPC upregulates DOR, and induces long-term protection either in cortical neurons against severe hypoxia, or in retina against elevated intraocular pressure (IOP) (Ma et al., 2005; Zhang et al., 2006; Peng et al., 2009). We therefore examined whether DOR is involved in the mediation of the neuroprotective effect induced by HPC in asphyxial CA rat model. In addition, it is well known that HPCinduced ischemia tolerance is profoundly dependent on hypoxia-inducible factor-1␣ (HIF-1␣) (Neubauer, 2001; Geocadin et al., 2005; Peng et al., 2009; Taie et al., 2009), then we further investigated the relationship between HIF-1␣ and DOR in the HPC-treated brain.
b
Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Center, Fourth Military Medical University, Xi’an, Shaanxi Province 710032, China
c
Department of Orthopaedics, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province 710038, China
Abstract—This study was designed to investigate whether delta opioid receptor (DOR) is involved in the neuroprotective effect induced by hypoxic preconditioning (HPC) in the asphyxial cardiac arrest (CA) rat model. Twenty-four hours after the end of 7-day HPC, the rats were subjected to 8-min asphyxiation and resuscitated with a standardized method. In the asphyxial CA rat model, HPC improved the neurological deficit score (NDS), inhibited neuronal apoptosis, and increased the number of viable hippocampal CA1 neurons at 24 h, 72 h, or 7 days after restoration of spontaneous circulation (ROSC); however, the above-mentioned neuroprotection of HPC was attenuated by naltrindole (a selective DOR antagonist). The expression of hypoxia-inducible factor-1␣ (HIF-1␣) and DOR, and the content of leucine enkephalin (L-ENK) in the brain were also investigated after the end of 7-day HPC. HPC upregulated the neuronal expression of HIF-1␣ and DOR, and synchronously elevated the content of L-ENK in the rat brain. HIF-1␣ siRNA was used to further elucidate the relationship between HIF-1␣ and DOR in the HPC-treated brain. Knockdown of HIF-1␣ by siRNA markedly abrogated the HPC induced upregulation of HIF-1␣ and DOR. The present study demonstrates that the expression of DOR in the rat brain is upregulated by HIF-1␣ following exposure to 7-day HPC, at the same time, HPC also increases the production of endogenous DOR ligand L-ENK in the brain. DOR activation after HPC results in prolonged neuroprotection against subsequent global cerebral ischemic injury, suggesting a new mechanism of HPC-induced neuroprotection on global cerebral ischemia following CA and resuscitation. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. 1
Contributed equally to this work. *Corresponding author. Tel: ⫹86-02984774501 or ⫹86-02984777439 or ⫹86-02984777539. E-mail address: [email protected] (Y.-Q. Li) or tdmzka@ fmmu.edu.cn (W. Chai) or [email protected] (X.-D. Sun). Abbreviations: ACSF, artificial cerebrospinal fluid; CA, cardiac arrest; CPR, cardiopulmonary resuscitation; Ct, threshold cycle; DADLE, [DAla2, D-Leu5] enkephalin; DOR, delta opioid receptor; HIF-1␣, hypoxiainducible factor-1␣; HPC, hypoxic preconditioning; L-ENK, leucine enkephalin; NDS, neurological deficit score; NTI, naltrindole; PB, phosphate buffer; PBS, phosphate buffered saline; ROSC, restoration of spontaneous circulation; RT-PCR, reverse transcription polymerase chain reaction; TBS, Tris-buffered saline; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling.
EXPERIMENTAL PROCEDURES Animal preparation This study was approved by the Animal Care and Use Committee of the Fourth Military Medical University (Xi’an, China), and followed the National Guidelines for Animal Experimentation. Male
0306-4522/12 $36.00 © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.11.060
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C.-J. Gao et al. / Neuroscience 202 (2012) 352–362 Sprague–Dawley rats weighing between 300 and 350 g were obtained from the experimental animal center of our university. Animals were individually housed and maintained on a 12 h light and dark cycle with free access to water and standard rat chow. Every effort was made to minimize animal suffering and reduce the number of animals used.
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ACSF⫹CA group and HPC⫹NTI⫹CA group received i.c.v. infusion of 10 l ACSF containing 0 or 50 nmol naltrindole 30 min before the induction of CA. The doses of naltrindole were determined according to our preliminary experiments. Survival rate after CA and CPR were calculated.
Neurological evaluation Induction of hypoxic preconditioning The day before the start of HPC, rats were acclimated to the custom-made Plexiglas chambers (one rat per chamber, 12⫻5⫻5 in.3, gas flow through chamber⫽4 L/min) under normoxic conditions. Within the chambers, O2 levels were continuously monitored with an S-3A Oxygen Analyzer (Applied Electrochemistry Inc., Sunnyvale, CA, USA). To induce HPC, rats were placed daily into the Plexiglas chambers in which oxygen content was reduced to 10% by continuously flushing with nitrogen from 0800 h– 0000 h daily for 7 days. Control animals were kept in the chambers under normoxic conditions. The animals were allowed free access to food and water during HPC.
Delta opioid receptor inhibition After anesthetizing by i.p. injection of 3% pentobarbital sodium (40 mg/kg), the rats were placed in a stereotaxic frame (SN-1C, Narishige Instruments, Tokyo, Japan) with a horizontal skull position. Then, 10 l artificial cerebrospinal fluid (ACSF) containing 50 nmol naltrindole (NTI, a selective DOR antagonist; Sigma, St Louis, MO, USA) was administered into the left cerebroventricle with a Hamilton microsyringe (80630, Hamilton Co., Reno, NV, USA), using the following coordinates: 0.8 mm posterior to bregma, 1.4 mm lateral to the midline on the left side, and 4.0 mm ventral to the skull surface. All coordinates were derived from according to the coordinates of the Paxinos and Watson atlas (Paxinos and Watson, 2004).
Cardiac arrest and cardiopulmonary resuscitation The procedure of CA and cardiopulmonary resuscitation (CPR) was performed as our previous study (Gao et al., 2010). Briefly, under anesthesia with 3% pentobarbital sodium (i.p. 40 mg/kg), each rat was intubated tracheally with a 14-gauge cannula and ventilated by a volume controlled small animal ventilator (DHX150, Medical Instrument Factory of Chengdu, China) with room air to maintain normocapnia. The arterial blood pressure (ABP) and electrocardiogram (ECG) were monitored. Asphyxia was induced by stopping mechanical ventilation and clamping the tracheal tubes at the end of expiration. After 8-min asphyxiation, CPR was initiated by administering a bolus injection of epinephrine (0.02 mg/kg, i.v.) and 5% sodium bicarbonate (1 mmol/kg, i.v.) followed by mechanical ventilation and thoracic compressions. Restoration of spontaneous circulation (ROSC) was defined as the return of supraventricular rhythm with an increase of mean artery pressure (MAP) beyond values of 60 mm Hg. Ventilation was consistently maintained until spontaneous breath started. Failure to restore spontaneous circulation within 10 min resulted in discontinuation of resuscitation efforts.
Experiment I To evaluate the effect of DOR inhibition on neuroprotection induced by HPC, rats were randomly assigned to CA, HPC⫹CA, NTI⫹CA, HPC⫹ACSF⫹CA, and HPC⫹NTI⫹CA groups (20 rats for each group). Animals in the CA group were only subjected to 8-min asphyxiation and CPR. Animals in NTI⫹CA group received ICV infusion of 50 nmol naltrindole 30 min before the induction of CA. All the rats in other three groups received 7-day HPC. At 24 h after the end of HPC, animals in HPC⫹CA group were subjected to 8-min asphyxiation and CPR, whereas animals in HPC⫹
To evaluate the neurological outcome in the successfully resuscitated animals (n⫽7 for each group), the neurological deficit score (NDS) was determined at 24 h, 72 h, and 7 days after ROSC according to previous studies (Geocadin et al., 2000; Jia et al., 2008). In brief, scoring includes seven parameters: general behavior, brain stem function, motor and sensory function assessment, motor behavior (including gait coordination and balance on beam), other behavior reflexes, and seizures. The NDS and its components are presented in Table 1. An NDS of 80 reflected Table 1. Neurological deficit Score (NDS) for rats (A) General behavioral Consciousness Arousal
Respiration
Total score⫽19 Normal [10], stuporous [5], comatose [0] Eyes open spontaneously [3], eyes open to pain [1], no eye opening [0] Normal [6], abnormal [0], absent [0]
(B) Brain-stem function Olfaction Vision Pupillary reflex Corneal reflex Startle reflex Whisker stimulation Swallowing
Total score⫽21 Present [3], absent Present [3], absent Present [3], absent Present [3], absent Present [3], absent Present [3], absent Present [3], absent
(C) Motor assessment Strength (left and right side tested and scored separately)
Total score⫽6 Normal [3], stiff/weak [1], no movement/paralyzed [0]
(D) Sensory assessment Pain (left and right side tested and scored separately)
Total score⫽6 Brisk withdrawal with pain [3], weak or abnormal response [1], no withdrawal [0]
(E) Motor behavior Gait coordination Balance on beam
Total score⫽6 Normal [3], abnormal [1], absent [0] Normal [3], abnormal [1], absent [0]
(F) Behavior Righting reflex Negative geotaxis Visual placing Turning alley
Total score⫽12 Normal [3], abnormal Normal [3], abnormal Normal [3], abnormal Normal [3], abnormal
(G) Seizures Convulsive or nonconvulsive
Total score⫽10 No seizure [10], focal seizure [5], general seizure [0]
[0] [0] [0] [0] [0] [0] [0]
[1], [1], [1], [1],
absent absent absent absent
[0] [0] [0] [0]
Balance beam testing is normal if the rat can cross a 2 cm wide by 1 m long beam suspended 0.5 m above the floor. Abnormal is scored if the rat attempts and does not continue or stays momentarily and falls. Absent is scored when the rat falls off immediately upon placement on the beam. Other behavior reflex subscores evaluated the following: (1) righting reflex (animal placed on its back is able to correct to upright position); (2) turning alley (the animal is made to walk and turn back at the end of a 15 cm⫻0.5 m alley); (3) visual placing (the animal is lifted and is able to visually orient itself to objects and depth); (4) negative geotaxis (animal placed on its back on a plane angled at 45° corrects itself and moves up the incline).
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normal brain function, whereas an NDS of 0 represented brain death. The evaluation was always carried out by the same investigator who was blinded to the experimental groups.
Histological examination At the 7th day after ROSC, the rats (n⫽7 for each group) were deeply anesthetized and transcardially perfused with 100 ml 0.9% saline followed by 500 ml 0.1 M phosphate buffer (PB, pH 7.4) that contained 4% paraformaldehyde and 2% picric acid. Brains were removed and post-fixed in the same fixative for 2– 4 h and then cryoprotected for 24 h at 4 °C in 0.1 M PB that contained 30% sucrose. Serial coronal sections (10 m) from the area located at 3.5 mm posterior to bregma were subsequently obtained using a Leica cryostat (Leica CM1800, Heidelberg, Germany) and stained with Cresyl Violet. The viable neurons in the hippocampal CA1 region were observed with an Olympus CH30 microscope (Olympus, Corporation; magnification 400-fold) attached to an Olympus DP70 digital microscope camera (Olympus America Inc., Melville, NY, USA). The viable neurons were considered to be those with visible nucleus and intact cytoplasm with discernable and rich Nissl staining. Neurons that had shrunken cell bodies with surrounding empty spaces were excluded. The viable neurons in the hippocampal CA1 region were observed with an Olympus CH30 microscope (Olympus, Corporation).
TUNEL staining Twenty-four hours after ROSC, samples from five groups (n⫽7 for each group) in Experiment I were used. The coronal sections were obtained as described in the histological examination experiment. Neuronal apoptosis in the hippocampus CA1 region was assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining using an in situ cell death detection kit (TMR Red, Roche, Mannheim, Germany) according to the manufacturer’s recommendations. TUNEL-positive neurons in the CA1 region were observed using the Olympus BX-60 fluorescence microscope (Olympus Corporation). As described by Yamashiro et al. (2007), the number of viable neurons and TUNEL-positive neurons in CA1 region were counted in the predefined area (0.25 mm2) at a magnification of 400-fold by an investigator blinded to the experimental groups, using AxioVision software (Zeiss, Jena, Germany).
Experiment II To determine the regulatory effect of HPC on the delta opioid system and HIF-1␣ before cardiac arrest, rats were randomly divided into two groups: Control group and HPC group. At 24 h after the end of HPC, three rats from each group were sacrificed for immunofluorescence staining. At 1 day, 3 days, or 7 days after the end of HPC, the brains were harvested for real-time reverse transcription polymerase chain reaction (RT-PCR), Western blot, or measurement of leucine enkephalin (n⫽5 for each time point).
Immunofluorescence staining The coronal sections were obtained as described in Experiment I. The 10 m thick coronal sections were incubated with primary antibodies: goat anti-DOR (1:100; Santa Cruz Biotechnology, CA, USA) and mouse anti-HIF-1␣ (1:100; Santa Cruz) for 48 h at 4 °C. The sections were washed three times in 0.01 M phosphate buffered saline (PBS; 10 min each) and then incubated for 6 h at room temperature with the secondary antibodies: FITC (fluorescein isothiocyanate)-labeled rabbit anti-goat IgG for DOR (1:200; Vector, Burlingame, CA, USA) and Cy3 (cyanin 3)-labeled donkey anti-mouse IgG for HIF-1␣ (1:200; Vector). After staining, the sections were then rinsed in PBS, air dried, coverslipped with a mixture of 50% glycerin and 2.5% triethylene diamine in PBS, and
observed with a confocal laser scanning microscope (Olympus FV1000, Tokyo, Japan).
Real-time RT-PCR Total RNA was extracted from the cortex and hippocampus using a Trizol kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was treated with RNase-free DNase I (Takara Biotechnology, Dalian, China) and reverse transcribed (RT) into cDNA with PrimeScript™ RT reagent Kit (Takara). The cDNA was then used as a template for real-time PCR with the primers for HIF-1␣: 5=-CCAGATTCAAGATCAGCCAGCA-3= (forward) and 5=-GCTGTCCACATCAAAGCAGTACTCA-3= (reverse), DOR: 5=-AACGTGCTCGTCATGTTTGG-3= (forward) and 5=-CAGGTACTTGGCGCTCTGGAA-3= (reverse), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase): 5=-GGCACAGTCAAGGCTGAGAATG-3= (forward) and 5=-ATGGTGGTGAAGACGCCAGTA-3= (reverse). Real-time RT-PCR reactions were performed in MiniOpticon Real-Time PCR Detection System (Bio-Rad Laboratories, CA, USA) using the SYBR Green RealTime PCR Master Mix (Toyobo Co. Ltd., Japan). PCR products were electrophoresed in 2% agarose gel (Invitrogen) to confirm that PCR yielded a single product of the expected size. The expected sizes were 100 bp (HIF-1␣), 133 bp (DOR), and 143 bp (GAPDH). The threshold cycle (Ct) was defined for each amplification plot. Each sample was run in triplicate and mean Ct values were used for further calculations. The results were analyzed by the 2⫺⌬⌬Ct method (Livak and Schmittgen, 2001). To ensure validity of our calculations, we confirmed that primer sets used in this study have the same efficiencies as ascertained by varying template concentrations. In each case, the log of the template concentration when plotted against ⌬Ct yielded values of less than 0.1 for the slope. The fold changes of mRNA levels in all samples were expressed relative to the calibrator (100%). The value of the control sample served as calibrator.
Western blot Western blot analysis was performed as described previously (Persson et al., 2005). Tissues from the cortex and hippocampus were homogenized for 20 min in lysis buffer containing Complete™ protease inhibitor cocktail (Santa Cruz Biotechnology) on ice. The samples were centrifuged for 25 min at 15,000 r/min at 4 °C. The supernatant containing total protein was collected, and the protein concentration was determined by Bradford method. The electrophoresis samples were heated at 100 °C for 5 min and loaded onto 10% SDS-polyacrylamide gel with standard Laemmli solutions (Bio-Rad Laboratories, CA, USA). The proteins were electroblotted onto polyvinylidene difluoride membranes (PVDF, Immobilon-P, Millipore, Billerica, MA, USA). The membranes were placed in a blocking solution, which contained Tris-buffered saline with 0.02% Tween (TBS-T) and 5% non-fat dry milk, for 1 h, and incubated overnight under gentle agitation with primary antibodies: goat anti-DOR (1:100; Santa Cruz), mouse anti-HIF-1␣ (1: 100; Santa Cruz), and rabbit anti--actin (1:5000; Sigma, St Louis, MO, USA). Bound primary antibodies were detected with the anti-goat, anti-mouse, or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). All reactions were detected by the enhanced chemiluminescence (ECL) detection method (Amersham). The densities of protein blots were analyzed by using Labworks Software (Ultra-Violet Products Ltd., Cambridge, UK) and normalized to -actin levels.
Measurement of leucine enkephalin The brain content of leucine enkephalin (L-ENK) was determined using a commercial kit (Phoenix Pharmaceuticals, Belmont, CA, USA) as described previously after peptide extraction (Ma et al., 2005). The values of L-ENK are expressed as ng/mg of protein.
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Experiment III To further elucidate the relationship between HIF-1␣ and DOR in the HPC-treated brain, rats were randomly divided into HIF-1␣ siRNA group and control siRNA group (n⫽5 for each group). At 24 h after the end of 7-day HPC, we performed in vivo HIF-1␣ siRNA transfer according to the method described previously (Chen et al., 2009). Then, 10 l HIF-1␣ siRNA (sc-45919, Santa Cruz) or control siRNA-A (sc-37007, Santa Cruz) was diluted with the same volume of transfection reagent (sc-29528, Santa Cruz) and mixed gently. After incubation for 45 min at room temperature, the mixture was injected intraparenchymally using a Hamilton microsyringe under the guidance of a stereotaxic frame (SN-1C) and the infusion was delivered over 60 min. The protein expressions of HIF-1␣ and DOR in the parietal cortex around the injection point were evaluated 24 h post-transfection using Western blot and immunofluorescence staining as described earlier in the text.
Statistical analysis The software, SPSS 13.0 for Windows (SPSS Inc., Chicago, USA), was used to conduct statistical analyses. All values, except for survival rate and neurological deficit score, are presented as mean⫾SD and were analyzed by one-way analysis of variance, and between-group differences were detected with post hoc Student–Newman–Keuls test. Survival rate comparison was done using the Fisher’s exact test. The neurological deficit scores were expressed as median (range) and analyzed with Kruskal–Wallis test followed by the Mann–Whitney U-test with Bonferroni correction. Values of P⬍0.05 were considered statistically significant.
RESULTS Survival rate The survival rate in the CA group was 75.0% (15/20 rats); in the HPC⫹CA group 90.0% (18/20 rats); in the NTI⫹CA group 70.0% (14/20 rats); in the HPC⫹ACSF⫹CA group 85.0% (17/20 rats); in the HPC⫹NTI⫹CA group 75.0% (15/20 rats). Statistical analysis revealed that there were no significant differences of the survival rate among all the groups using the Fisher exact test. Data collected from rats that did not survive were not included in the following analysis. Physiological variables and characteristics Before induction of CA, there were no differences in body weight, temperature, arterial blood gas, and hemodynamic variables among all the groups (data not shown). Parameters related to the resuscitation procedures, such as MAP, heart rate, and arterial blood gas data during CPR and the early ROSC phase did not differ in the five groups (data not shown). HPC-induced improvement of neurological outcome is reversed by naltrindole The NDS was determined at 24 h, 72 h, and 7 days after ROSC and the results are shown in Fig. 1. There were significant differences among the five groups with regard to the neurological status at all the three time points after ROSC (P⬍0.05). Compared with the CA group, the ani-
Fig. 1. Neurological deficit score (NDS) was presented as median and 25th–75th percentile (n⫽8). Significant differences were observed among the five groups with regard to the neurological status at all the three time points after ROSC. HPC significantly improved the neurological deficit scores, whereas the DOR antagonist naltrindole reversed the beneficial effect of HPC. * P⬍0.01 versus CA group; # P⬍0.01 versus HPC⫹CA group.
mals in the HPC⫹CA group had persistently better functional recovery in NDS at all time points with statistical significance (P⬍0.01). The DOR antagonist naltrindole had no effect on NDS when administered alone but reversed the beneficial effect of HPC given 30 min before the onset of CA (P⬍0.01, HPC⫹NTI⫹CA versus HPC⫹CA). The NDS of the HPC⫹ACSF⫹CA group was similar to that of the HPC⫹CA group. There were no statistical differences in NDS among the CA, NTI⫹CA, and HPC⫹ NTI⫹CA groups. HPC-induced improvement of morphology changes is abolished by naltrindole Compared with the sham animal, histological evaluation of the hippocampal CA1 region revealed various degrees of neuronal damage in all the surviving animals following CA and CPR (Fig. 2A). However, the number of viable neurons in the hippocampal CA1 region was significantly increased in the HPC⫹CA group in comparison with the CA group as revealed by Nissl staining (P⬍0.01, Fig. 2B); there was no difference between the HPC⫹ACSF⫹CA and HPC⫹CA groups. The number of viable neurons in the NTI⫹CA group was similar to that in the CA group. Naltrindole reduced the number of viable neurons in the HPC⫹ NTI⫹CA group compared with the HPC⫹CA group (P⬍0.05, Fig. 2B). HPC-induced reduction of neuronal apoptosis is attenuated by naltrindole No positive TUNEL staining (red) neuron was detected in the brain sections of sham animals. However, a large number of TUNEL-positive neurons in the hippocampal CA1 region of rat brain were seen in the CA, NTI⫹CA, and HPC⫹NTI⫹CA groups, whereas in contrast, only a small amount of TUNEL-positive neurons in the HPC⫹ACSF⫹
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Fig. 2. (A) Representative photomicrographs (Nissl staining; magnification 400-fold) showing the viable neurons in hippocampal CA1 region at the 7th day after ROSC. (B) The number of viable neurons in the hippocampal CA1 region detected through Nissl staining. HPC significantly increased the number of viable neurons, whereas the DOR antagonist naltrindole abolished the protective effect of HPC. * P⬍0.01 versus CA group; # P⬍0.05 versus HPC⫹CA group. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
CA and HPC⫹CA groups were observed (Fig. 3A). The quantitative analysis of the number of TUNEL-positive neurons in the hippocampal CA1 region showed that HPC significantly reduced the number of TUNEL-positive neurons compared with the CA and NTI⫹CA groups (P⬍0.01). Naltrindole reversed the beneficial effect of HPC given 30 min before the onset of CA (P⬍0.05, HPC⫹NTI⫹CA versus HPC⫹CA, Fig. 3B). HPC upregulates the expression of HIF-1␣ and DOR in the brain The changes in the mRNA and protein levels of HIF-1␣ were paralleled in groups (Figs. 4A and 5A). The expression of HIF-1␣ mRNA and protein in both the cortex and hippocampus significantly increased at 1 day and 3 days after the end of 7-day HPC as compared with those in the control groups (Figs. 4B and 5B, P⬍0.05), but returned to normal level at 7 days after the end of 7-day HPC. HPC also produced evident upregulation of DOR expression in the brain as revealed by the presence of both mRNA (Fig. 4A) and protein (Fig. 5A). The expression of DOR mRNA
and protein in the hippocampus and cortex significantly increased at 1 day and 3 days after the end of 7-day HPC as compared with those in the control group (Figs. 4C and 5C, P⬍0.05), but returned to normal level at 7 days after the end of 7-day HPC. The expression of HIF-1␣ and DOR was also examined by immunofluorescence staining. As shown in Fig. 6, in the control group, little expression of HIF-1␣ was observed in the hippocampal CA1, CA3, and cortex of rat brain. However, the expression of HIF-1␣ was significantly increased after 7-day HPC induction. Compared with the control animal, DOR was mostly upregulated after 7-day HPC induction in hippocampal CA1, CA3, and cortex of rat brain. HPC increases the content of L-ENK in brain tissue Fig. 7 showed that endogenous opioid L-ENK was present in the control brain tissue. The content of L-ENK was significantly increased in the HPC-treated rat brains at 1 day and 3 days after the end of 7-day HPC; however, its
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Fig. 3. Neuronal apoptosis in hippocampal CA1 region at 24 h after ROSC. (A) Representative photomicrographs of TUNEL staining (magnification 400-fold). (B) Quantitative analysis of the number of TUNEL-positive neurons. HPC significantly decreased the number of TUNEL-positive neurons, whereas the DOR antagonist naltrindole attenuated the reduction in the number of TUNEL-positive neurons. * P⬍0.01 versus CA group; # P⬍0.05 versus HPC⫹CA group. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
level was slightly but insignificantly decreased at 7 days after the end of 7-day HPC. Knockdown of HIF-1␣ by siRNA decreases the expression of HIF-1␣ and DOR Twenty-four hours after intraparenchymal administration of HIF-1␣ siRNA or control siRNA-A, HIF-1␣ and DOR protein in rat brain were analyzed by Western blot. The protein expression of HIF-1␣ in the parietal cortex around the injection point was significantly reduced in 7-day HPC rats that were treated with HIF-1␣ siRNA when compared with the control siRNA-A. Interestingly, the expression of DOR was also decreased by HIF-1␣ siRNA (Fig. 8, P⬍0.05). The expression of HIF-1␣ and DOR was also examined by immunofluorescence staining, and the results were consistent with Western blot experiments (Fig. 9).
DISCUSSION Recently, DOR has been reported to participate in the mechanisms of endogenous neuroprotective effects of
HPC in cultured cortical neurons as well as in rat retina in vivo (Ma et al., 2005; Zhang et al., 2006; Peng et al., 2009). However, little is known about the involvement of the DOR in HPC-induced global cerebral ischemic tolerance. Asphyxial CA is one of the most important causes of global cerebral ischemia. Therefore, we used a rat model of asphyxial CA to observe neuroprotective effects of HPC and investigate the role of DOR in HPC-induced neuroprotection. The HPC protocol (10% O2, 4 h/day for 7 days) used in this study induced that kind of delayed tolerance and exerted potent neuroprotection manifested as inhibition of neuronal apoptosis and alleviation in hippocampus CA1 neuron injury as well as amelioration of neurological outcome in our asphyxial CA model. Using this asphyxial CA rat model, we demonstrated that neuroprotective effects of HPC were attenuated by a highly selective DOR antagonist, naltrindole. The results indicate that DOR plays important roles in HPC-induced neuroprotection on global cerebral ischemic injury following CA and CPR.
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lated by 5% oxygen as shown in Ma’s work (Ma et al., 2005). This conception is also supported by previous in vivo studies. Peng and colleagues have also shown that 4 weeks of mild hypoxic upregulates DOR expression in rat retinas (Peng et al., 2009). In the present study, we found that the upregulation of DOR mRNA and protein in the brain was synchronous after 7-day HPC. Both of DOR mRNA and protein expression increased at 1 day after the end of HPC and maintained at least for 3 days, then returned to normal level at 7 days after the end of HPC. Another main hypothesis tested in this study is that the content of endogenous DOR ligand in the brain would be modulated by 7-day HPC. It is interesting to note that HPC produced an elevation of the content of L-ENK in the brain tissue. Not only that, this elevated L-ENK could be maintained for at least 3 days after 7-day HPC, which was consistent with the upregulation of DOR mRNA and protein. As is well known, an
Fig. 4. The mRNA expression of HIF-1␣ and DOR was analyzed with real-time RT-polymerase chain reaction. The PCR products were demonstrated by Panel (A). Panel (B, C) showed HPC significantly upregulated the mRNA expression of HIF-1␣ and DOR in both hippocampus and cortex at 1 d and 3 d after the end of 7-d HPC. * P⬍0.05 versus Control group.
Preconditioning research is an attractive experimental strategy to identify endogenous protective mechanisms. The rapid tolerance of HPC is independent of protein synthesis and mediated by posttranslational modification, whereas the delayed effects of HPC appears to require synthesis of new RNA and protein (Barone et al., 1998; Sharp et al., 2004). An important finding of the present work was that HPC induced upregulation of the protein and mRNA expression of DOR in the hippocampus and cortex. DOR is an oxygen-sensitive protein, and DOR expression varies greatly, depending on the duration and extent of hypoxia (Ma et al., 2005; Zhang et al., 2006). Besides downregulation after prolonged and severe hypoxia, DOR expression in the cultured cortical neurons was upregu-
Fig. 5. The protein expression of HIF-1␣ and DOR was evaluated with Western blot. Representative immunoblots of HIF-1␣ and DOR were demonstrated by Panel (A). Panel (B, C) showed HPC significantly upregulated the protein expression of HIF-1␣ and DOR in both hippocampus and cortex at 1 d and 3 d after the end of 7-d HPC. * P⬍0.05 versus Control group.
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Fig. 6. Representative immunofluorescence staining for HIF-1␣ and DOR in hippocampal CA1, CA3, and cortex of rat brain at 24 h after 7-d HPC. Both HIF-1␣ and DOR expression were mostly upregulated in the HPC group compared with the Control group. Scale bar⫽20 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
increase in receptor agonists may increase receptor-mediated intracellular activity. The number of receptors may
be another major rate-limiting factor in terms of the increase in receptor-mediated activity. Thus, we can con-
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Fig. 7. Changes of leucine enkephalin in the hippocampus and cortex. HPC significantly increased the content of leucine enkephalin in the brain at 1 d and 3 d after the end of 7-d HPC. * P⬍0.05 versus Control group.
clude from the present study that HPC increases the expression of DOR and the production of the endogenous DOR ligand L-ENK, and the ligand-receptor reaction elicits protective effects against global cerebral ischemic injury following CA and CPR. Since DOR belongs to the G protein-coupled receptor family, G protein activity along with its downstream signaling pathway may be involved in the initial steps of HPC-induced neuroprotection. It has been deduced that the DOR-G protein-PKC-pERK-Bcl 2 pathway is one of the most important neuroprotective mechanisms in cortical neurons and can be activated by HPC to counteract stress-induced death signals (Ma et al., 2005). Recently, Peng and colleagues demonstrated that neuroprotective effect of HPC against IOP elevation involves the restoration of redox imbalance and the inhibition of pro-apoptotic caspase 3 after DOR-ERK activation (Peng et al., 2009). This notion provided novel insights into the mechanism of DOR-mediated HPC neuroprotection. However, the pathophysiologic responses to global cerebral
ischemia induced by asphyxial CA are inherently different from all the above situations. We believe that further studies are needed to elucidate the intracellular mechanisms underlying DOR-mediated HPC neuroprotection in global cerebral ischemia following CA. HIF-1␣ is an important transcription protein that regulates gene expression in the brain and other tissues in response to decreases in oxygen availability. Large amounts of data have demonstrated that HIF-1␣ is essential for the induction of tolerance to ischemia by HPC. It is well known that HPC induces the expression of the protein HIF-1␣ and its downstream genes such as vascular endothelial growth factor (VEGF), erythropoietin (EPO), inducible nitric oxide synthase (iNOS), and so on. However, several studies (Ma et al., 2005; Zhang et al., 2006; Peng et al., 2009) and our results indicated that DOR protein was involved in the neuroprotection of HPC. We also observed that HPC synchronously upregulated the expression of HIF-1␣ and DOR in the brain. So we further investigated the relationship between HIF-1␣ and DOR in the HPC-treated brain. An important finding was that HIF-1␣ siRNA markedly abrogated the HPC-induced upregulation of HIF-1␣ and DOR. The previously mentioned results strongly indicated that there was a close relationship between HIF-1␣ and DOR in the HPC-treated brain, and DOR may be another downstream gene of HIF-1␣. Peng et al. have also observed the similar relationship between HIF-1␣ and DOR in the HPC-treated retina (Peng et al., 2009). However, further studies are needed to reveal the precise interaction between HIF-1␣ and DOR.
CONCLUSIONS In conclusion, the expression of DOR in the rat brain is upregulated by HIF-1␣ following exposure to 7-day HPC, at the same time, HPC also increases the production of endogenous DOR ligand L-ENK in the brain. DOR activation after HPC results in prolonged neuroprotection against subsequent global cerebral ischemic injury, suggesting a
Fig. 8. Knockdown of HIF-1␣ by siRNA decreases the expression of HIF-1␣ and DOR in the parietal cortex around the injection point. Representative immunoblots of HIF-1␣ and DOR were demonstrated by Panel (A). Panel (B) showed HIF-1␣ siRNA markedly abrogated the HPC-induced upregulation of HIF-1␣ and DOR proteins. * P⬍0.05 versus Control siRNA group.
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Fig. 9. Representative immunofluorescence staining for HIF-1␣ and DOR in the parietal cortex around the injection point at 24 h post-transfection. Both HIF-1␣ and DOR expression were mostly downregulated in the HIF-1␣ siRNA group compared with the Control siRNA group. Scale bar⫽20 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
new mechanism of HPC-induced neuroprotection on global cerebral ischemia following CA and CPR. Acknowledgments—This work was supported by the National Natural Science Foundation of China (Grant. 81071527) and the Chinese Postdoctoral Science Foundation (Grant. 201003743).
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(Accepted 28 November 2011) (Available online 21 December 2011)