Apocynin attenuate spatial learning deficits and oxidative responses to intermittent hypoxia

Sleep Medicine 11 (2010) 205–212

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Original Article

Apocynin attenuate spatial learning deficits and oxidative responses to intermittent hypoxia Liu Hui-guo *, Liu Kui, Zhou Yan-ning, Xu Yong-jian Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Respiratory Disease, Ministry of Health, Wuhan 430030, China

a r t i c l e

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Article history: Received 11 January 2009 Received in revised form 8 May 2009 Accepted 16 May 2009 Available online 18 January 2010 Keywords: Apocynin Intermittent hypoxia Spatial learning NADPH oxidase Oxidative stress P22phox P47phox

a b s t r a c t Rationale: The long-term intermittent hypoxia (LTIH) that characterizes sleep-disordered breathing impairs spatial learning and increases oxidative stress in rodents. We hypothesized that LTIH activated brain NADPH oxidase, which served as a critical source of superoxide in the oxidation injury, and that apocynin might attenuate LTIH-induced spatial learning deficits by reducing LTIH-induced NADPH oxidase expression. Objective: To investigate the effects of apocynin on spatial learning and oxidative responses to LTIH in rats. Methods: Forty healthy male Sprague–Dawley (SD) rats were randomly divided into four groups of 10 each: a LTIH group, an apocynin-treated LTIH group, a sham LTIH group and an apocynin-treated sham group. Spatial learning in each group was assessed with the Morris water maze test. RT-PCR and Western blot were used to examine mRNA and protein expression of NADPH oxidase subunit p47phox and p22phox in the hippocampus region. The level of MDA and SOD were detected by colorimetric method. The terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL) method was used to display the apoptotic cells of the hippocampus tissue. Results: Apocynin treatment prevented LTIH-induced decreases in spatial learning during the Morris water maze as well as LTIH-induced decrease in SOD levels. In untreated animals, LTIH exposure was related to increase of MDA levels in comparison to sham LTIH animals, and apocynin-treated animal exposure to LTIH showed reduction in MDA levels. Increases in hippocampus NADPH oxidase subunit p47phox mRNA and protein expression were observed in LTIH-exposed animals; there was no statistical difference of p47phox mRNA and protein expression between LTIH group and apocynin treatment group. Treatment with apocynin significantly ameliorated cell apoptosis in LTIH-exposed animals. Conclusion: These results indicate that apocynin attenuates LTIH-induced spatial learning deficits and mitigates LTIH-induced oxidative stress through multiple beneficial effects on oxidant pathways. NADPH oxidase up-expression is closely associated with oxidative processes in LTIH rats, and inhibition of NADPH oxidase activity may hopefully serve as a useful strategy for cognitive function impairment from chronic intermittent hypoxia. Ó 2009 Elsevier B.V. All rights reserved.

1. Introduction Over the last 20 years, a substantial body of evidence has accumulated to indicate that sleep-disordered breathing (SDB) is a growing health problem in both adult and pediatric populations [1–3]. Young et al. [4] suggested that as much as 2–4% of the general population in developed countries may suffer from the most common form of SDB, obstructive sleep apnea (OSA). The clinical syndrome of OSA is characterized by repeated episodes of upper airway obstruction during sleep and substantial neuropsychological impairments in humans [5]. Chronic exposure to intermittent hypoxia as encountered in OSA is marked by neurodegenerative

* Corresponding author. Address: Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, #1095 Jiefang Road, Wuhan 430030, China. Tel./fax: +86 27 83747135. E-mail address: [email protected] (L. Hui-guo). 1389-9457/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.sleep.2009.05.015

changes in the adult rat brain [6]; most notably are increases in programmed cell death in CA1 of the hippocampus and adjacent cortex as well as impaired spatial learning in the Morris water maze [7,8]. The sensitivity of the hippocampus to hypoxic insults and its well established role as a critical structure for learning and memory suggest that exposure to intermittent hypoxia may play a significant role in the cognitive disturbances seen in patients with OSA [9]. Oxidative stress in association with spatial learning impairments in rats has been implicated in the pathophysiological mechanisms underlying several neurodegenerative brain disorders and enhances neuronal susceptibility to glutamate excitotoxicity [10–13]. Hypoxia and ischemia are related to increased oxygen radical production and membrane lipid peroxidation [14–17], suggesting that oxidative stress is a potential contributor to the cellular injury and behavioral impairments associated with intermittent hypoxia.

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NADPH oxidase-dependent production of superoxide radical has been identified as a major contributor to oxidative injury in the brain under conditions of both inflammation and severe hypoxia/reperfusion injury [18–21]. Moreover, NADPH oxidase has been implicated in oxidative neurodegeneration, including Alzheimer’s disease [22,23], and in dopaminergic neuronal injury in murine models of Parkinson’s disease [24–26]. Several recent reports have identified NADPH oxidase in select populations of neurons [27–29], raising the possibility that neuronal NADPH oxidase activation could contribute to enhanced neuronal vulnerability to oxidative injury. Presently, it is unknown whether intermittent hypoxia that models sleep apnea increases NADPH oxidase in the region of the hippocampus which is a critical structure for learning and memory, or whether NADPH oxidase might mediate the intermittent hypoxia-induced oxidative injury, and/or proinflammatory responses. Apocynin, 1-(4-hdroxy-3-methoxyphenyl) ethanone, is a plantderived drug, discovered during activity-guided isolation of immunomodulatory constituents from Picrorhiza kurroa. The generation of reactive oxygen species (ROS) by serum-treated zymosan-triggered human neutrophils from healthy blood donors was inhibited by apocynin in a dose-dependent manner (50% inhibition at 68 Um) [30,31]. The mode of action may involve (myeloperoxidase-dependent) metabolization and inhibition of NADPH assembly by interfering with the intracellular translocation of two cytosolic components, p47phox and p67phox [32]. In this study, we first determined whether hypoxia/reoxygenation events in adult rats that modeled severe sleep apnea oxygenation increased the expression of p47phox and p22phox gene which were common components of all types of NADPH oxidases and played an essential role in NADPH oxidase activation [33,34]. We then determined the impact of apocynin on the expression of p22phox and p47phox, the levels of MDA and SOD which were commonly used as indicators of oxidative stress, and spatial learning impairments in our animal model of SDB. 2. Methods 2.1. Animals Eight-week-old male Sprague–Dawley (SD) rats (150–200 g; n = 40; The Laboratory of Respiratory Disease of Tongji Medical College, Wuhan, China) were used in this study. Animals were housed in groups of three in clear polycarbonate cages (55  45  35 cm) with food and water available ad libitum and were confirmed pathogen free at the time of study. Rats were randomly divided into four groups of 10 each: a LTIH group, an apocynin-treated LTIH group, a sham LTIH group and an apocynin-treated sham group. The animal use protocol listed below had been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Tongji Medical College of Huazhong University of Science and Technology. 2.2. Long-term intermittent hypoxia protocol The LTIH protocol details were published recently [6]. Briefly, an automated nitrogen/oxygen profile system was used to produce brief reductions in ambient oxygen level from 21% to 10% for 5s every 90s for LTIH, resulting in arterial oxyhemoglobin saturation fluctuations between 95–98% and 83–86%. Sham LTIH, with ambient FIO2 fluctuations from 21% to 20% for 5s every 90s, held arterial oxyhemoglobin values constant between 96% and 98%. To determine if NADPH oxidase inhibition would prevent LTIH gene responses, apocynin (Sigma) was intragastrically administered (3 mg/kg/day in 50 ll of 0.9% saline) to 10 rats for 3 days prior

and throughout intermittent hypoxia exposure. Both LTIH and sham LTIH conditions were produced for 10 h of the lights-on period for a total of 4 weeks. Humidity, ambient CO2, and environmental temperature were held constant. 2.3. Behavioral testing with the Morris water maze Spatial learning was assessed in a Morris water maze as described previously [7,35]. Behavioral testing consisted of a standard place-training reference memory task in the water maze. Rats were trained to locate a hidden, submerged platform while only using distal spatial cues. The rats were placed in a pool (180 cm in diameter, filled to a depth of 50 cm, and maintained at 23 ± 1 °C) and had to find a platform hidden below the water surface (made opaque with paint) using visual cues in the room which remained constant. For each trial, the rat was placed into the pool from quasirandom start points (N, S, E, or W) and allowed a maximum of 60 s to escape the platform, where it was allowed to remain for 30 s. Mean latencies were analyzed. Spatial probe was conducted after the completion of place-training and used as an indicator of spatial bias. Upon completion on the place-training, the platform was removed, the percentage of time spent in quadrant and the number of passes over the previous target platform in 2 min were analyzed. The Ethovision system by Noldus Information Technology was used to quantify Morris water maze performance. 2.4. Oxidative stress Oxidative stress is a well-established mechanism of cellular injury in the brain. Superoxide dismutase (SOD) and malondialdehyde (MDA), the commonly used indicator of oxidative stress, lipid peroxidation, and subsequent cellular injury in cells and tissues were assayed using SOD kits and MDA kits according to instructions (Nanjing ‘‘JianCheng” Institute of Biological Engineering, China). Briefly, after anesthesia with pentobarbital (50 mg/kg intraperitoneally), the hippocampus of laboratory rats were dissected, snap frozen in liquid nitrogen, and stored at 80 °C until assay the next day. Hippocampus tissues were homogenized in 20 mM phosphate buffer (pH 7.4) containing 0.5 mM butylated hydroxytoluene to prevent sample oxidation. After proteins concentration measurement, the samples were then measured at 550 nm. The level of SOD and MDA were calculated with the standard curve according to the manufacturer’s instructions (Nanjing ‘‘JianCheng” Institute of Biological Engineering, China). 2.5. RT-PCR for P47phox and P22phox mRNA expression Total RNA from hippocampus was extracted using Trizol reagent (Sigma) according to the instructions. To check the integrity of the total RNA, 1 lg was fractionated on a 2% agarose gel. RNA concentration was quantified spectrophotometrically and had a 280/260 optical density ratio between 1.8 and 2.0. After extraction of total RNA, 2 lg were reverse transcribed to cDNA using reverse transcription reagents (TOYOBO, Japan). The PCR primers were designed by Shanghai Sangon Biological Engineering Technology Corporation. The primers used for p47phox gene expression were 50 GGG TGG TCA GGA AAG GG 30 and 50 GCG GAG TCG ATG GAT TG 30 . The PCR mixture contained 100 ng genomic DNA template, 0.2 lmol/L of each primer, 0.8 mmol/L of each deoxynucleoside triphosphate, 1.5 U Taq polymerase (Fermentas Corporation), and reaction buffer in a total volume of 25 ll. PCR was performed for 35 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 57 °C, and extension for 1 min at 72 °C, with initial denaturation at 95 °C for 10 min, and a final extension at 72 °C for 10 min. Amplification resulted in a 279-bp fragment. The primers used

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2.6. Western blotting for P47phox protein expression For analysis of p47phox protein, hippocampal samples were homogenized in lysis buffer containing 50 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 0.5% deoxycholic acid, 1% NP40, 0.1% SDS, 1 mmol/L PMSF, and 100 mg/L leupeptin and were centrifuged at 12,000 rpm for 15 min at 4 °C; supernatants were harvested. Protein concentrations were measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, USA). Of the total protein, 75-lg was separated on 10% SDS–polyacrylamide gels and transferred onto a nitrocellulose membrane (Pierce, USA). Before immunostaining, the membranes were blocked with 5% non-fat milk overnight at 4 °C, followed by incubation with appropriate dilutions of the primary specific antibody, Rabbit p47phox antibody (1:1000, Santa Cruz Biotechnology Inc., USA), in PBST at 4 °C for 16 h. The secondary antibody was horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (1:10,000, Santa Cruz Biotechnology Inc., USA) incubation was carried out at room temperature for 1 h. b-Actin was probed with anti-b-actin IgG as a loading control. The membranes were then treated with an enhanced chemiluminescence reagent (Amersham, USA), and the signals were detected by exposure of the membranes to X-ray films (Kodak, USA). The relative signal intensity was quantified by densitometry with Gel pro3.0 image software (Media Cybernetics, Silverspring, MD, USA). The protein expression was represented as the optical density ratio of the interest protein in comparison to that of b-actin. All experiments were performed three times independently. 2.7. TUNEL staining in the hippocampus Animals (n = 4 for each groups) were fixed by cardiac perfusion using 4% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer. After perfusion fixation, the brains were immersion-fixed in the same fixative overnight at 4 °C. Forty lm thick vibratome sections were cut. TUNEL staining for apoptotic neurons in the hippocampus was performed according to instructions of the manufacturer (Boster Biological Technology, Ltd., China). In brief, tissue sections were fixed in 4% paraformaldehyde in PBS, pH 7.4, for 10 min at room temperature, post-fixed in precooled ethanol:acetic acid (2:1, v/v) for 5 min at 20 °C, and treated with 3% hydrogen peroxide to quench endogenous peroxidase activity. After adding the equilibration buffer, sections were treated with terminal deoxynucleotidyl transferase (TdT) and digoxigenin-dNTPs for 60 min at 37 °C. Specimens were then treated with anti-digoxigenin-peroxidase for 30 min at 37 °C, colorized with 3,30 -diaminobenzidine (DAB) substrate, and counterstained with 0.5% methyl green. Finally, slides were rinsed, dehydrated, and mounted. A negative control was prepared by omitting the TdT enzyme to control for non-specific incorporation of nucleotides or binding of enzyme-conjugate. The specimens were examined using a bright-field microscope (Zeiss Axioskop; Carl Zeiss GmbH, Jena, Germany) and the data expressed as the number of TUNEL-positive cells/high-power field (200) in at least five high-power fields.

2.8. Statistical date analysis All statistical analysis was calculated using SPSS version 13.0. Descriptive characteristics of group variables were expressed as means ± SD. The significance of variables between groups was tested by analysis of variance (ANOVA), Student–Newman–Keuls post hoc tests were used when appropriate. P values <0.05 were considered significant.

3. Results The results of the behavior testing were summarized in Figs. 1–3. As compared with the sham LTIH rats (21.32 ± 4.44), the mean latencies in LTIH rats (52.20 ± 6.09) were longer significantly (P < 0.05); mean latencies were significantly lower in apocynin treatment LTIH rats (38.05 ± 5.12) than those in LTIH rats (P < 0.05). No significant differences were observed between sham LTIH rats and apocynin-treated sham LTIH rats (20.55 ± 4.30) (P = 0.76). As compared with sham LTIH rats (4.16 ± 1.47), the numbers of passes over the previous target platform in LTIH rats (0.83 ± 0.75) were reduced (P < 0.05); the numbers of passed over the previous target platform were significantly higher in apocynin treatment rats (2.33 ± 0.82) than those in LTIH rats (P < 0.05). No significant differences were observed between sham LTIH rats and apocynin-treated sham LTIH rats (3.83 ± 1.60) (P = 0.64). As compared with sham LTIH rats (39.34 ± 6.19), the percentage of time spent in quadrant in LTIH rats (18.06 ± 4.34) was reduced (P < 0.05); the percentage of time spent in quadrant was significantly higher in apocynin treatment rats (30.35 ± 3.50) than those in LTIH groups (P < 0.05). No significant differences were observed between sham LTIH rats and apocynin-treated sham LTIH rats (38.52 ± 3.15) (P = 0.75). Fig. 4 showed the average MDA concentrations in hippocampus region from LTIH and sham LTIH animals receiving vehicle or apocynin. LTIH groups [2.35 ± 0.15 (mol/mgprot)] were associated with significant increase in the relative MDA production in the hippocampus of rats in comparison to sham LTIH groups [0.97 ± 0.16 (mol/mgprot)] (P < 0.05); the amount of MDA production in animals exposed to 4 weeks of intermittent hypoxia treated with the apocynin [1.73 ± 0.18 (mol/mgprot)] was significantly lower than LTIH groups (P < 0.05). Animals exposed to apocynin-treated sham LTIH [0.75 ± 0.18 (mol/mgprot)] showed reductions in MDA levels compared with sham LTIH rats (P < 0.05). Fig. 5 showed

70 60 Escape latency (s)

for p22phox gene expression were 50 CGC CGT GGT GAA GCT GTT CG 30 and 50 CTG GGC GGC TGC TTG ATG GT 30 . The housekeeping GAPDH PCR products were used as an internal control; their sequences were 50 TGC TGT CCC TGT ATG CCT CT 30 and 50 GGT CTT TAC GGA TGT CAA CG 30 . The PCR and condition were the same as p47phox except for annealing at 62 °C and 55 °C, respectively. Amplification resulted in a 237-bp fragment and 462-bp fragment, respectively. PCR products were run in 2% agarose gels (Invitrogen) along with 100 base-pair (bp) ladder markers. Amplified products were visualized by staining with ethidium bromide and were analyzed using gel scanner, and the ration of p22phox/GAPDH was determined and used to compare groups.

50 40 30 20 10 0 Sham LTIH

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Apocynintreated LTIH

Fig. 1. Mean latencies (s) to locate the target platform during place-training in rats exposed to long-term intermittent hypoxia (LTIH) and sham LTIH receiving vehicle or apocynin. LTIH rats displayed significantly longer mean latencies for the target platform in comparison to sham LTIH rats (P < 0.05). A significant decrease of mean latencies was observed in animals receiving apocynin during LTIH (P < 0.05). No significant differences were observed between sham LTIH rats and apocynintreated sham LTIH rats (P = 0.76).

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Fig. 2. The numbers of passes over the previous target platform during spatial probe performance in rats exposed to long-term intermittent hypoxia (LTIH) and sham LTIH receiving vehicle or apocynin. As compared with sham LTIH rats, the numbers of passes over the previous target platform in LTIH groups were reduced (P < 0.05). A significantly increase of numbers of passes over the target platform were observed in animals receiving apocynin during LTIH (P < 0.05). No significant differences were observed between sham LTIH rats and apocynin-treated sham LTIH rats (P = 0.64).

)

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Fig. 5. Relative superoxide dismutase (SOD) production, one of most important components of the antioxidant defense system, in the hippocampus of rats exposed to sham LTIH or LTIH and treated with apocynin or vehicle. As compared with sham LTIH rats, the SOD levels in LTIH groups were reduced (P < 0.05). A significant increase in SOD levels was observed in apocynin treatment groups in comparison to LTIH groups (P < 0.05). No significant differences were observed between sham LTIH rats and apocynin-treated sham LTIH rats (P = 0.34).

significant increase in SOD levels was observed in apocynin treatment groups [81.17 ± 10.28 (U/mgprot)] in comparison to LTIH groups (P < 0.05). No significant differences were observed between sham LTIH rats and apocynin-treated sham LTIH rats [105.67 ± 17.36 (U/mgprot)] (P = 0.34). Gene expression of the p47phox and p22phox subunit of NADPH oxidase was measured in hippocampus regions using

50 45 40 35 30 25 20 15 10 5 0

1 Sham LTIH

Apocynin-treated sham LTIH

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Fig. 3. The percentage of time spent in quadrant during spatial probe performance in rats exposed to long-term intermittent hypoxia (LTIH) and sham LTIH receiving vehicle or apocynin. As compared with sham LTIH rats, the percentage of time spent in quadrant in LTIH groups was reduced (P < 0.05). A significantly increase of percentage in target quadrant were observed in animals receiving apocynin during LTIH (P < 0.05). No significant differences were observed between sham LTIH rats and apocynin-treated sham LTIH rats (P = 0.75).

M bp

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Fig. 4. Relative malondialdehyde (MDA) production, an indicator of lipid peroxidation, in the hippocampus of rats exposed to sham LTIH or LTIH and treated with apocynin or vehicle. As compared with sham LTIH rats, the MDA levels in LTIH groups were increased (P < 0.05). The amount of MDA production in animals exposed to 4 weeks of intermittent hypoxia treated with the apocynin was significantly lower than LTIH groups (P < 0.05). Animals exposed to apocynintreated sham LTIH showed reductions in MDA levels compared with sham LTIH rats (P < 0.05).

the average SOD concentrations in the hippocampus region from LTIH and sham LTIH animals receiving vehicle or apocynin. LTIH group [65.14 ± 14.18 (U/mgprot)] showed lower SOD levels compared with sham LTIH groups [98.17 ± 9.13 (U/mgprot)]; a

P47phox/GAPDH ratio in hippocampus

MDA (mol/mgprot)

Apocynin-treated LTIH

3

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0 Sham LTIH

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sham LTIH

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Fig. 6. (Upper panel) Representative pattern of gene expression of p47phox and GAPDH in sham LTIH (lane 1), apocynin-treated sham LTIH (lane 2), LTIH (lane 3), and apocynin-treated LTIH (lane 4). Lane M shows the DNA ladder (100–600 bp). (Lower panel) Mean ratios of p47phox to GAPDH in hippocampus from rats exposed to sham LTIH or LTIH and treated with apocynin or vehicle (#P < 0.05 versus sham LTIH; NP = 0.41 versus LTIH; *P = 0.34 versus sham LTIH). The result is reported as means ± SD, P < 0.05 was considered statistically significant.

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M

1

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(1.68 ± 0.29; 1.73 ± 0.24, respectively) (P < 0.05). This elevation of p47phox and p22phox was not attenuated in animals receiving apocynin during LTIH (2.05 ± 0.11; 2.01 ± 0.12, respectively) (P = 0.41; P = 0.38), such that no significant differences emerged in sham LTIH animals apocynin-treated sham LTIH animals (1.55 ± 0.23; 1.62 ± 0.15, respectively) (P = 0.34; P = 0.31) (Figs. 6 and 7). P47phox protein expression was determined in hippocampus via Western blotting. The ratio of p47phox/b-actin and p22phox/ b-actin within the hippocampus region were elevated in LTIH rats (0.56 ± 0.10) in comparison to sham LTIH rats (0.28 ± 0.12) (P < 0.05). This elevation of p47phox was not attenuated in animals receiving apocynin during LTIH (0.53 ± 0.14) (P = 0.62), such that no significant differences emerged in sham LTIH animals or apocynin-treated sham LTIH animals (0.27 ± 0.10) (P = 0.81) (Fig. 8). TUNEL staining method was used to evaluate apoptosis in the hippocampus. The results indicated that neuron apoptosis indices in the hippocampus region were elevated in LTIH rats (0.52 ± 0.13) in comparison to sham LTIH rats (0.14 ± 0.03) (P < 0.05). This elevation of apoptosis indices was attenuated in animals receiving apocynin during LTIH (0.29 ± 0.03) (P < 0.05) (Fig. 9).

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Fig. 7. (Upper panel) Representative pattern of gene expression of p22phox and GAPDH in sham LTIH (lane 1), apocynin-treated sham LTIH (lane 2), LTIH (lane 3), and apocynin-treated LTIH (lane 4). Lane M shows the DNA ladder (100–600 bp). (Lower panel) Mean ratios of p22phox to GAPDH in hippocampus from rats exposed to sham LTIH or LTIH and treated with apocynin or vehicle (#P < 0.05 versus sham LTIH; NP = 0.38 versus LTIH; *P = 0.31 versus sham LTIH). The result is reported as means ± SD, P < 0.05 was considered statistically significant.

RT-PCR. The ratio of p47phox/GAPDH and p22phox/GAPDH within the hippocampus region was elevated in LTIH rats (2.17 ± 0.28; 2.12 ± 0.23, respectively) in comparison to sham LTIH rats

During the last two decades, OSA has increasingly been recognized as a serious and frequent health condition with potential long-term morbidities that include learning and psychological disabilities, metabolic consequences, and increased severity and prevalence of cardiovascular disorders [36–39]. The hippocampus and prefrontal cortex, critical structures for learning and memory, are particularly sensitive to the hypoxic events occurring during extended periods of episodic hypoxia during sleep, and these changes lead to significant cognitive deficits in the rodent [8,40]. In addition, a growing body of evidence suggests that the adverse neurobehavioral consequences imposed by IH stem, at least in part, resulted from activation of oxidative stress

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0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Sham LTIH

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Fig. 8. (Upper panels) Representative Western blots for p47phox and b-actin in hippocampus from rats exposed to sham LTIH or LTIH and treated with apocynin or vehicle. (Lower panel) Mean ratios of p47phox to b-actin in hippocampus from rats exposed to sham LTIH or LTIH and treated with apocynin or vehicle (#P < 0.05 versus sham LTIH; N P = 0.62 versus LTIH; *P = 0.81 versus sham LTIH). The result is reported as means ± SD, P < 0.05 was considered statistically significant.

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a

Apoptosis indices in the hippocampus

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Fig. 9. (Upper panels) TUNEL staining in Sham LTIH (a); LTIH (b); Apocynin-treated LTIH (c). (Lower panel) Apoptosis indices in hippocampus from rats exposed to sham LTIH or LTIH and treated with apocynin (#P < 0.05 versus sham LTIH; NP < 0.05 versus LTIH). The result is reported as means ± SD, P < 0.05 was considered statistically significant.

and inflammatory signaling cascades. Up-regulation of genes that play important roles in the induction and maintenance of inflammatory processes, such as cytokines and cyclooxygenases (COX), as well as increased eicosanoid and platelet-activating factor (PAF)-mediated neuroinflammation have been demonstrated after exposure to IH in rodents [6,41–45]. Such changes can amplify nitric oxide-induced cellular injury via oxidative and nitrosative pathways, most likely through nuclear factor-Kb-dependent pathways [46,47]. The collective actions of such oxidative and inflammatory processes likely compromise cell viability within sensitive brain regions while failing to appropriately recruit inducible defense processes, resulting in increased neuronal vulnerability and functional deficits. Although the causal relationship between inflammatory and oxidative pathways remains to be elucidated, the available data suggest that reductions in oxidative stress may offer great potential therapeutic utility in animal models of sleep apnea and in patients suffering from the disease [6,41– 45] . Oxidative stress is a well-established mechanism of cellular injury in the brain because brain tissue contains a large amount of polysaturated fatty acids, which are highly susceptible to oxidative reactions. Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and are easy to decompose to form a complex series of compounds. They include reactive carbonyl compounds, of which the most abundant is MDA, a commonly used indicator of lipid peroxidation, oxidative stress, and subsequent cellular injury in cells and tissues. In our experiments, we observed that LTIH was associated with significant increases in MDA in the hippocampus, and this elevation was attenuated in animals receiving apocynin treatment. SOD is one of most important components of the antioxidant defense system. According to previous findings [48], decreased SOD activity might contribute to free radical production. The enhanced ROS production might lead to the lower SOD activity. In our experiments, the level of SOD in LTIH group was lower than that of control group. We also observed that IH-induced decrease in SOD was attenuated by apocynin administration. The mechanism by which LTIH triggers decrease of SOD is unknown. NADPH-oxidase-dependent production of superoxide radical has been identified as a major contributor to oxidative injury

in the brain under conditions of severe hypoxia and inflammation [49]. NADPH oxidase has also been implicated in neurodegeneration, such as Alzheimer’s and Parkinson’s disease, and NADPH oxidase is increasingly recognized for its role in health and disease, necessary for normal immunity and cell signaling [49]. Long-term exposure to frequent hypoxia/reoxygenation events that mimic the altered oxygenation patterns of SDB has been shown to induce NADPH oxidase in selected brain regions, suggesting that activation of this enzyme may partly underlie the increased neuronal inflammation and oxidative stress observed in animal models of SDB [43]. Our findings provide further support for the hypothesis that intermittent hypoxia increases NADPH oxidase subunit p47phox and p22phox mRNA expression in hypoxia sensitive brain regions involved in learning and memory. Apocynin administration was not able to attenuate the increase in NADPH oxidase subunit p47phox and p22phox gene expression under IH conditions. These findings are consistent with previous reports that systemic NADPH oxidase activity inhibition via apocynin was realized by inhibiting the aggregation of NADPH oxidase subunits formed [50]. Our Western blot studies confirmed significant increases in levels of p47phox expression during the period of LTIH in the hippocampus. Collectively, these findings highlight the significance of long-term hypoxia/reoxygenation events as in sleep apnea and identify a novel pathway whereby the hypoxia/reoxygenation events induce NADPH oxidase activation in the brain. Therefore, these findings identify a potential target pathway for prevention of neurobehavioral morbidities commonly observed in persons treated for OSA. Exposure to LTIH, such as occurred in patients with sleep apnea, is associated with marked impairments of hippocampus-dependent learning tasks, and these deficits are attenuated via reductions in oxidative stress and inflammatory signaling cascades through pharmacological interventions [42,46]. Similarly, on spatial probe trials administered after completing place-training, apocynin-treated rats exposed to LTIH displayed significantly greater spatial bias for the previous hidden platform position, indicating that apocynin was capable of attenuating LTIH-induced spatial learning deficits, presumably through reductions of the

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degenerative oxidative and inflammatory mechanisms associated with LTIH; this might represent a potential interventional strategy for patients with SDB. Our data gathered all show that oxidative injury in the hippocampus is associated with increased expression of NADPH oxidase and that inhibition of NADPH oxidase prevents spatial learning impairments. Nevertheless the mechanism by which LTIH triggers activation of NADPH oxidase is unknown. Observations associating NADPH oxidase subunit with oxygen-sensing processes in a variety of mammalian cells suggested that the enzyme functions as an oxygen sensor to produce reactive oxygen species in response to changes in oxygen tension [51]. Further study is needed to determine the mechanism by which LTIH triggers NADPH oxidase activity in the brain. In summary, we report novel findings on the effect of apocynin in the context of intermittent hypoxia during sleep, a condition with markedly important clinical relevance, considering the high prevalence of SDB. We herein show that apocynin administration produces significant reductions in the induction of pathophysiological mechanisms implicated in the neuronal damage associated with IH and that parallel improvements in IH-induced hippocampus-dependent deficits occur with apocynin intake. Although the rodent model simulates the oxygenation pattern of moderate to severe SDB, it does not incorporate other potentially injurious elements of SDB, such as sleep fragmentation or recurring hypercapnia. Notwithstanding such considerations, our present findings indicate that apocynin will attenuate oxidative stress and inflammatory mechanisms underlying IH-induced end-organ damage in the rat brain. These novel findings clearly warrant further research aimed at defining the roles of apocynin as potential pharmacologic supplements in the prevention of SDB-induced neurocognitive morbidities.

Acknowledgments We are grateful to Wu Huai-ming for statistical advice. This work was supported by Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.

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