Neurochem Res DOI 10.1007/s11064-015-1586-1
ORIGINAL PAPER
Intermittent Hypoxia-Induced Parvalbumin-Immunoreactive Interneurons Loss and Neurobehavioral Impairment is Mediated by NADPH-Oxidase-2 Liang Yuan1 • Jing Wu1 • Jiang Liu2 • Guowei Li1 • Dong Liang3
Received: 23 December 2014 / Revised: 21 March 2015 / Accepted: 20 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Obstructive sleep apnea usually contribute to psychiatric diseases and cognitive impairments in adults. Loss of parvalbumin (PV)-immunoreactive interneurons (PV-IN) in the brain cortex is an important feature of psychiatric diseases, such as schizophrenia. Here we investigate the causal contribution of oxidative stress in the brain cortex to neuropathological alterations in a mouse model of sleep apnea. Wild-type (WT) and the NADPHoxidase-2 (gp91-phox/NOX2) knock-out adult male C57BL/6J mice were exposed to intermittent hypoxia (IH) or standard room air in the same chamber. In vivo we determined the impact (1) of IH exposures on NOX2 expression, (2) of genetic gp91-phox/NOX2 knock-out and (3) of pharmacological NOX2 inhibition on IH-induced neuropathological alterations in adult mice. Endpoints were oxidative stress, PV-IN and neurobehavioral alterations. The results showed IH exposures increased NOX2 expression in the prefrontal cortex of WT mice, which was accompanied with elevations of indirect markers of oxidative stress (HNE, HIF-1a, 8-OHDG). WT mice showed
Liang Yuan and Jing Wu have equally contributed to this work. & Dong Liang [email protected] 1
Department of Anaesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, People’s Republic of China
2
Comprehensive Cancer Center of Drum-Tower Hospital, School of Medicine, Nanjing University, Nanjing, People’s Republic of China
3
Department of Emergency, Ruijin Hospital North, School of Medicine, Shanghai Jiaotong University, 999 Xiwang Road, Jiading District, Shanghai 201801, People’s Republic of China
loss of PV-IN in the prefrontal cortex and increased locomotion activity and anxiety levels after exposed to IH, while no change emerged in NOX2 knock-out mice. Treatment of WT mice with the antioxidant/NOX inhibitor apocynin prevented the neuropathological and neurobehavioral alterations induced by IH exposures. Our data suggest that NOX2-derived oxidative stress is involved in the loss of PV-IN in the prefrontal cortex and development of neurobehavioral alterations for adult mice exposed to IH. These results provide a molecular mechanism for the coupling between sleep apnea and brain oxidative stress as well as potential new therapeutic avenues. Keywords Intermittent hypoxia Oxidative stress NADPH-oxidase-2 PV-immunoreactive interneurons Neurobehavioral alterations
Introduction Obstructive sleep apnea (OSA), a clinical syndrome that is characterized by repeated episodes of upper airway obstruction during sleep, is a significant and highly prevalent health problem, due not only to its cardiovascular and metabolic morbidity, but also because of the prominent cognitive and behavioral implications of the disease. The neuropsychological impairments are accompanied by increased levels of systemic markers of oxidative stress and inflammation in nervous system [1, 2]. The inordinate sensitivity of neuronal tissues to alterations in oxygen homeostasis has led to the hypothesis that the neuropathological and neurobehavioral alterations observed in OSA patients are associated, at least in part, with the episodic hypoxia-reoxygenation cycles during sleep that characterize OSA. In support of this hypothesis, animal
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models have demonstrated that chronic exposure to IH is accompanied with neurodegenerative changes, increased oxidative stress, and impaired spatial learning and memory in the morris water maze [3–6]. Genetic or pharmacological manipulations of oxidative stress pathways attenuated IH-induced deficits in neuropathological and neurobehavioral alterations. NADPH oxidase is composed of catalytic and regulatory subunits which include two membrane-bound subunits (gp91-phox and p22-phox) and three cytosolic subunits, which include p47-phox, p67-phox, and Rac [7]. The membrane-bound subunits form a heterodimer that stabilizes them within the membrane, whereas the cytosolic subunits are recruited to the membrane following stimulation. Complete complex assembly is necessary for full NADPH oxidase activity [8]. Neurons express the NOX2 isoform of NADPH oxidase, which contains the gp91-phox catalytic subunit and requires the p47-phox assembly subunit [9]. In neurons, NOX2 enzyme is thought to be involved in cell fate and modulation of neuronal activity [10]. From a pathologic point of view, NOX enzymes are involved in the increase of oxidative stress seen in a variety of brain disorders, from psychiatric to neurodegenerative diseases [11, 12]. Cortical interneurons are classified into several subtypes that contribute to cortical oscillatory activity. PV-expressing cells, a type of inhibitory interneuron, are involved in the gamma oscillations of local field potentials (LFPs). PV-expressing cells are the major type of inhibitory gammaaminobutyric acid-producing (GABAergic) interneurons of the neocortex. These cells are distributed across cortical layers, target the proximal regions of pyramidal cells, and are characterized by a fast-spiking physiological phenotype. Loss of phenotype of PV-IN in the brain cortex and psychiatric behavioral alterations are often reported in the previous studies [13, 14], which are regarded as the important features of psychiatric diseases, such as schizophrenia [15–17]. Exposure to IH, a hallmark of OSA, is associated with increased oxidative stress in the central nervous system (CNS) and neurobehavioral impairments. HIF-1a (hypoxia-inducible factor-1a) is an indirect marker for oxidative stress, excessive NADPH oxidase activity and HIF1a expression has been shown in IH-induced neuropathological alterations [18–20]. HIF-1a is required for IH-induced reactive oxygen species (ROS) generation and plays a pro-oxidant role in the setting of IH exposure [18]. In the present study, we proposed loss of PV-IN in the brain was an important feature of IH-induced neuropathological alterations. By using multiple pharmacological and genetic approaches, we evaluate the possible role of oxidative stress in the development of neuropathological and neurobehavioral alterations induced by IH.
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Materials and Methods Animals NADPH-oxidase-2 knock-out (KO) C57BL/6J mice were originally obtained from Jackson Laboratories and were bred with wild-type (WT) C57BL/6J mice at the Animal Center of Jinling Hospital, Nanjing, China, with free access to food and water. Littermate KO and WT animals were bred from heterozygote (±) females and KO (0/-) males resulting in WT (0/?) and KO (0/-) male littermates. Genotyping of animals used in the experiment was performed as previously described [8]. The primers (Cyber-gene) showed either the WT (50 -AAGAGAAACTCCTCTGCTGTGAA-30 ) with a band of 240 bp or the KO (5-GTTCTAATTCCATCAG AAGCTTATCG-30 ) with a band of 195 bp in addition to the common band (50 -CGCACT GGAACCCCTGAGAAA GG-30 ). The PCR reaction consisted of 12 cycles: denaturation: 94 °C, 20 s; annealing: 64 °C, 30 s; elongation: 72 °C, 35 s followed by an additional 25 cycles: denaturation: 94 °C, 20 s; annealing: 58 °C, 30 s; elongation: 72 °C, 35 s. The PCR products were analyzed by using TBE agarose electrophoresis (1.5 %), labeled with ethidium bromide. The animal experiments were approved by the Ethics Committee of Jin-ling Hospital, Nanjing University.
Experimental Design of the Study The experimental design were as follows (see Fig. 9b): in the first set of experiments: the authors determined the impact of IH on NOX2 expression and neuropathological alterations in the WT mice. The apocynin was used to determine the effect of pharmacological NOX2 inhibition on IH-induced neuropathological alterations. For the experiment, WT mice were treated either with apocynin (APO, Sigma, St. Louis, MO, USA; 4 mg/kg) or vehicle (VEH: 0.5 % DMSO) by intraperitoneal (i.p.) injection 30 min before and immediately after the exposure to 8 h daily for 14 days of IH. In the second set of experiments: the authors determined the effect of genetically NOX2 KO in mice on IH-induced neuropathological alterations. In the third set of experiments: the authors determined the effect of digoxin (D, HIF-1a inhibitor) on IH-induced NOX2 expression in the WT mice and investigated whether IH-induced expression of NOX2 was mediated by HIF-1a. For the experiment, WT mice were treated either with digoxin (Sigma, St. Louis, MO, USA; 1 mg/ kg, i.p.) or vehicle (VEH: 0.5 % DMSO, i.p.) 30 min before and immediately after the exposure to 8 h daily for 14 days of IH exposures.
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Intermittent Hypoxia Exposures WT (0/?) and KO (0/-) male (body weight, 20–25 g) housed in feeding cages were exposed to IH for 8 h per day for 14 days as previously described (IH group) [18]. Briefly, mice were placed in a specialized chamber, which was flushed with alternating cycles of pure nitrogen and compressed standard room air (SA). The IH profile consisted of alternating SA and nitrogen every 90 s. During hypoxia, inspired O2 levels rapidly reached a nadir of 5 % O2. Oxygen concentration was continuously measured by an O2 analyzer, and was changed by a computerized system controlling gas outlets. Control experiments were exposed to alternating cycles flushing SA every 90 s instead of nitrogen. Behavioral Testing For study of IH-induced neuropathological and psychiatric alterations, behavioral alterations in open-field test, elevated plus maze (EPM), forced swimming test (FST), fear conditioning test were carried out consecutively after IH exposure. The movement of each mouse was monitored and analyzed using a computer-operated video tracking system. The apparatus was cleaned after each trial. All apparatus used in tests were come from Shanghai Soft maze Information Technology Co, Ltd, China. All mice after behavioral tests were not used for any biochemistry studies and would be administered euthanasia. Open Field Emotional responses to the novel environment were measured by an open-field test. The open field task approaches the conflict between the innate fear that rodents have of the central area of a novel or brightly lit open field versus their desire to explore new environments. When anxious, the natural tendency of rodents is to prefer staying close to the walls (thigmotaxis). In this context, anxiety-related behavior is measured by the degree to which mice avoid the center of the open field test. Mice were placed in an openfield (40 9 40 9 40 cm) chamber made of white acrylic for 10 min under 70 lux lighting conditions, in which the video tracking system quantifies the number of entries into and time spent in the center of the locomotion arena. Activity was measured as the total distance traveled. Elevated Plus Maze (EPM) The EPM was used to assess anxiety. The basic measure is the animal preference for dark, enclosed places over bright, exposed places. Mice prefer to enter into closed arms compared to open arms. Time spent in the dark area is viewed as avoidance or anxiety-like behavior. Mice were
placed in the center of the maze facing a closed arm, and allowed to explore for 10 min in isolation. The following parameters were scored: (a) percent time spent in open arms and number of entries to open arms; (b) total number of entries to arms; (c) the number of stretched-attend postures (SAPs), defined as a posture in which the subject stretches forward and then retracts to its original position, performed from the central platform or enclosed-arms towards open-arms, was also recorded. This latter response is categorized as risk assessment, and has also been considered closed related to anxiety [21]. Fear Conditioning Fear conditioning was performed in a black plastic chamber (XR-XC404) equipped with a stainless steel grid floor. The conditioning (acquisition) trial for contextual and cued fear conditioning consisted of a 2 min exploration period followed by three conditioned stimulus (CS)–unconditioned stimulus (US) pairings separated by 1 min each (US, 0.70 mA footshock intensity, 2 s duration; CS, 70 db white noise, 20 s duration; US was delivered during the last 2 s of the CS presentation). A contextual test was performed in the conditioning chamber for 3 min in the absence of white noise at 48 h after conditioning. A cued test (for the same set of mice) was performed by presentation of the cue (70 db white noise, 3 min duration) in an alternative context with distinct visual and tactile cues. The cued test was conducted after the contextual test was finished. The rate of freezing response of mice was used to measure the fear memory. The level of nonspecific freezing provoked by the new context was controlled for 2 min before the presentation of the cue in that new context. Brain Tissue Harvest and Protein Level Quantification Different groups of mice which have not been subjected to behavior tests were used for biochemistry studies after IH exposure. In brief, the mice were anesthetized by sodium pentobarbital (50 mg/kg) followed by transcardial perfusion with paraformaldehyde 4 % in phosphate buffered saline (PBS; pH 7.35) for immunohistochemistry studies or PBS for western blot studies. For the western blot analysis, the harvested brain tissues were homogenized on ice using immunoprecipitation buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, and 0.5 % Nonidet P-40) plus protease inhibitors (1 lg/ml aprotinin, 1 lg/ml leupeptin, and 1 lg/ml pepstatin A) as described in previous studies. The lysates were collected, centrifuged at 12,000 rpm for 10 min, and quantified for total protein with the bicinchoninic acid protein assay kit (Pierce Technology Co., Iselin, NJ, USA).
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Western Blot Analysis The samples were subjected to western blot analysis according to the standard procedure using antibodies for hypoxia-inducible factor 1a (HIF-1a) (1:500, #sc-10790, Santa Cruz Biotechnology, Santa Cruz, CA, USA), NOX2 (1:2000, #ab129068, Abcam, Cambridge, MA, USA), p47phox (1:1000, #sc-7660, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and b-actin (1:10,000, Sigma, St. Louis, MO, USA). Quantitation of bands was undertaken using the Image J software (NIH Image, USA). We quantified the western blots bands by using b-actin levels to normalize protein levels (e.g., determining the ratio of NOX2 to b-actin amount) and control for loading differences in the total protein amount. Immunohistochemistry PV-IN in the prefrontal cortex were evaluated by immunohistochemical staining after IH exposure. The immunohistochemistry was performed according to the standard procedure. Every six sections were used for staining. The sections were deparaffinized, washed and incubated with PV antibody (1:1000; #mab1572; Millipore) and biotinylated secondary antibody. The PV-positive cells in the mouse cortex were counted manually in five randomly selected areas under a 20 9 objective microscope by an investigator who was blinded to the experiments. Six brains from each group were used for immunohistochemistry alalysis, and six brain sections of 5 lm thickness were examined in each brain. Data are given as mean number of cells/mm2. Measurement of NADPH Oxidase Activity The prefrontal cortex tissues were isolated and homogenized in a RIPA buffer. The homogenate was subjected to centrifugation at 250g for 10 min to remove cellular debris. Supernatant was then centrifuged at 20,000g for 20 min at 4 °C to eliminate mitochondria, lysosomes, peroxisomes, Golgi membranes, and rough endoplasmic reticulum. The resulting supernatant was centrifuged at 100,000g for 60 min at 4 °C. The pelleted plasma membrane fraction containing NOX was dissolved in a buffer containing 8 mM piperazine-N, N’-bis 2-ethanesulfonic acid (pH 7.2), 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, and proteolytic inhibitors. The solubilized membrane fraction samples were placed in a multiwell plate. NOX activity was measured using a cytochrome c reduction assay [18]. Briefly, 100 lg aliquots of membrane proteins were incubated in 25 mM HEPES buffer (pH 7.0) with 150 lm cytochrome c, 100 lm NADPH for 30 min at 37 °C in the presence and
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absence of superoxide dismutase (SOD; 200 U/ml). Cytochrome c reduction was measured by determining absorbance at 550 nm. The amount of SOD-inhibitable NOX activity expressed in nmol/min/mg protein was calculated using an extinction coefficient of 21 mM-1cm-1. Measurement of Lipid Peroxidation in the Prefrontal Cortex Since the most abundant lipid peroxidation product is 4-hydroxy-2-nonenal (HNE), measurement of this product has been used as an indicator of lipid peroxidation. The samples were subjected to analysis as previously described [22]. The prefrontal cortex was used for measurement of HNE using the bioxytech HAE-586 spectrophotometric assay kit (OxisResearch, Portland, OR, USA). The HNE concentrations in experimental samples were determined against HNE standards provided in the assay kit. Measurement of 8-Hydroxydeoxyguanosine (8-OHDG) The mice were anesthetized by sodium pentobarbital (50 mg/ kg) followed by transcardial perfusion with phosphate buffered saline (PBS; pH 7.35) for 5 min and the prefrontal cortex was dissected, snap frozen in liquid nitrogen, and stored at –80 °C until assay the following day. Cortical tissues were homogenized in 20 mM phosphate buffer (pH 7.4) containing 0.5 mM butylated hydroxytoluene to prevent sample oxidation. Levels of 8-OHDG (an oxidized form of deoxyguanosine that is a widely used biomarker of in vivo oxidative DNA damage) were measured in the prefrontal cortex using a commercially available assay (Cell Biolabs, San Diego, CA, USA) [20]. Briefly, cortical samples or 8-OHDG standards were first added to an 8-OHDG/BSA conjugate preabsorbed enzyme immunoassay plate. After a brief incubation, an anti—8-OHDG mAb was added, followed by a horseradish peroxidase-conjugated secondary antibody. The 8-OHDG content in the cortical samples was then determined by comparison with the 8-OHDG standard curve. Statistical Analysis Data regarding biochemistry changes and behavioral tests were expressed as mean ± SEM and analyzed by the Statistical Product for Social Sciences (SPSS; version 16.0, IL, USA). Normal distribution of data was analyzed using the Kolmogorov–Smirnov test. The data were performed using the student two-sample t test or one-way ANOVA followed by the post hoc Bonferroni test. When data did not fulfill parametric assumptions, data were expressed as medians and interquartile ranges and were analyzed using nonparametric tests (Kruskall–Wallis test with Dunn’s
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multiple comparisons test). In all experiments, differences were considered statistically significant at p B 0.05.
NOX-related oxidative stress in the loss of PV immunoreactivity induced by IH exposures.
Results
IH Increased HIF-a Expression in the Prefrontal Cortex
IH Exposures Induced Loss of PV-IN in the Prefrontal Cortex The authors determined alterations of the number of PV-IN in the prefrontal cortex to study the effect of IH exposures on brain neuropathological alterations and investigated the relationship between NOX2-dependent oxidative stress and the decrease in PV immunoreactivity. The results showed that IH exposures in mice induced loss of PV-IN in the prefrontal cortex compared with SA, which was attenuated by pre-treatment with apocynin before IH exposures or in gp91-phox/NOX2 KO mice (Fig. 1; F3,23 = 14.763, p \ 0.05, one-way ANOVA followed by the post hoc Bonferroni test). These findings suggest a possible role of
In accordance with the previous studies [18], we showed IH increased HIF-a expression in the prefrontal cortex (Fig. 2; p = 0.002, Kruskall–Wallis test with Dunn’s multiple comparisons test). In the present study, using multiple pharmacological and genetic approaches, the increased HIF-a expression was attenuated in gp91-phox/ NOX2 KO mice or by pretreatment with either the antioxidant/NOX inhibitor apocynin or HIF-1a inhibitor digoxin (p \ 0.05, Kruskall–Wallis test with Dunn’s multiple comparisons test), suggesting IH activated HIF-1a in a NOX2-dependent manner. HIF-1a Mediated the Increase of NOX2 Expression and Activity in Response to IH The results (Figs. 3a, 4) demonstrated IH exposures led to a substantial increase in NOX2 expression and activity in the prefrontal cortex in WT mice (p \ 0.01, Kruskall–Wallis test with Dunn’s multiple comparisons test). Such IH-induced increase of NOX2 expression and activity were much attenuated in gp91-phox/NOX2 KO mice and by pretreatment of antioxidant/NOX inhibitor apocynin
Fig. 1 IH exposures induced loss of PV-IN in the prefrontal cortex, which was attenuated in gp91-phox/NOX2 knock-out mice or pretreatment with apocynin before IH exposures. a Representative images of PV-IN in the prefrontal cortex. (original magnification 9200). b Quantification of the immunohistochemistry image. Data are given as mean number of cells/mm2. (mean ± SEM; n = 6; **p B 0.01 versus SA group; #p B 0.05 versus IH group)
Fig. 2 IH exposures induced HIF-1a expression in the prefrontal cortex of WT mice, which was prevented by HIF-1a inhibitor digoxin and the antioxidant/NOX inhibitor apocynin. Gp91-phox/NOX2 knock-out in mice also prevented the IH-induced HIF-1a expression in the prefrontal cortex of mice. a Representative western blot images of HIF-1a protein in the prefrontal cortex of all groups of mice to illustrate band intensities. b Histogram showed results of the densitometric analysis of western blot images of HIF-1a. (mean ± SEM; n = 6; **p B 0.01 vs. SA group; #p B 0.05, ##p B 0.01 vs. IH group)
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Fig. 3 IH exposures induced NOX2 expression in the prefrontal cortex of WT mice which was prevented by HIF-1a inhibitor digoxin. Gp91-phox/NOX2 knock-out in mice completely prevented the IHinduced NOX2 expression in the prefrontal cortex of mice. a Western blot showed HIF-1a inhibitor digoxin prevented the IH-induced NOX2 expression in the prefrontal cortex of WT mice. Lack of the
NOX2 band in the brain proteins extracted from the gp91-phox/ NOX2 knock-out mice (far right lane) confirmed specificity of the anti-NOX2 antibody and location of NOX2 specific band. b Western showed upregulation of the cytosolic subunits p47-phox after IH exposures. (mean ± SEM; n = 6; **p \ 0.01, ***p B 0.001 vs. SA group; ##p B 0.01 vs. IH group)
Indirect Markers of Oxidative Stress (HNE, 8-OHDG) were Increased After IH Exposures in the Prefrontal Cortex of WT Mice
Fig. 4 NADPH oxidase activity in the prefrontal cortex was measured as NADPH-dependent cytochrome c reduction. IH exposures resulted in a substantial increase in NOX activity in WT mice. Such IH-induced increase of NADPH oxidase activity were much attenuated in gp91-phox/NOX2 knock-out mice and by pretreatment of HIF-1a inhibitor digoxin and antioxidant/NOX inhibitor apocynin. (mean ± SEM; n = 6; **p B 0.01 vs. SA group; #p B 0.05, ## p B 0.01 vs. IH group)
(p \ 0.05, Kruskall–Wallis test with Dunn’s multiple comparisons test). To investigate whether HIF-1 is required for IH-induced NOX2 expression and activity, a pharmacological approach targeting inhibiting HIF-1a protein synthesis [18] was employed. The results showed the IHincreased NOX2 expression and activity in the prefrontal cortex of WT mice was prevented by HIF-1a inhibitor digoxin (p \ 0.05, Kruskall–Wallis test with Dunn’s multiple comparisons test). Lack of the NOX2 band in brain proteins extracted from a gp91-phox/NOX2 KO mice confirmed specificity of the anti-Nox2 antibody (Fig. 3a). IH exposures upregulated the cytosolic subunits p47-phox which was necessary for full NADPH oxidase activity (Fig. 3b; p = 0.007, Student’s t test).
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In the present study, the authors showed IH exposures induced indirect markers of oxidative stress (HNE, 8-OHDG) in the prefrontal contex of WT mice (HNE: Fig. 5a; p = 0.002, Kruskall–Wallis test with Dunn’s multiple comparisons test; 8-OHDG: Fig. 5b; F4,29 = 13.775, p = 0.006, one-way ANOVA followed by the post hoc Bonferroni test). To test whether the signs of oxidative stress detected in the brain of IH-exposed mice could be prevented pharmacologically or genetically, the antioxidant/NOX inhibitor apocynin, HIF-1a inhibitor digoxin and genetically gp91phox/NOX2 KO in mice was employed. Results showed that the administration of digoxin, apocynin and genetically gp91-phox/NOX2 KO in mice prevented IH-induced lipid peroxidation (Fig. 5a) and DNA oxidation (Fig. 5b) in the prefrontal contex after IH exposures (p \ 0.05). IH Exposures Increased the Spontaneous Locomotion Activity and Anxiety-Related Behavior in Mice To correlate molecular changes with behavioral observations and investigate the relationship between NOX2-dependent oxidative stress and behavioral alterations in mice, emotional responses and habituation to the novel environment were measured by an open-field test for 10 min. Open field is a sensorimotor test for locomotion, habituation, exploratory, and emotional behavior, including risk assessment and anxiety-like behavior, in novel
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Fig. 5 IH exposures induced lipid peroxidation and DNA oxidation in the prefrontal contex. The administration of HIF-1a inhibitor digoxin, antioxidant/NOX inhibitor apocynin and gp91-phox/NOX2
knock-out in mice prevented IH-induced a lipid peroxidation and b DNA oxidation in the prefrontal contex. (mean ± SEM; n = 6; **p B 0.01 vs. SA group; #p B 0.05, ##p B 0.01 vs. IH group)
environments. The open field was performed with a focus on behavioral functions that are physiologically regulated by the prefrontal cortex, such as spontaneous locomotion activity. Behavioral tests showed an increase in spontaneous locomotion activity (Fig. 6a; F3,36 = 7.681, p = 0.006, one-way ANOVA followed by the post hoc Bonferroni test) and a decrease of time in centre and number of center entries in IH-exposed mice compared with SA-exposed mice (Fig. 6b; F3,39 = 9.425, p = 0.027; Fig. 6c; F3,39 = 11.594, p = 0.031; one-way ANOVA followed by the post hoc Bonferroni test). The antioxidant/ NOX inhibitor apocynin administration and genetically gp91-phox/NOX2 KO in mice attenuated the IH-induced increase in spontaneous locomotion activity (Fig. 6a; p \ 0.05) and decrease of time in centre (Fig. 6b; p \ 0.05) and number of center entries (Fig. 6c; p \ 0.05). The results suggested IH exposures increased the spontaneous locomotion activity and anxiety-related behavior in mice and might be associated with further development of psychiatric diseases.
NOX2-dependent oxidative stress and anxiety in mice, we performed the EPM test which is used to assess anxiety. The authors showed WT mice exposed to IH spent significantly less time in the open arm of the EPM compared to SA, IH ? APO and IH ? KO mice (Fig. 7a; F3,39 = 4.885, p \ 0.05, one-way ANOVA followed by the post hoc Bonferroni test). The number of entries into the open-arms was also significantly decreased in IH-exposed WT mice (Fig. 7b; F3,39 = 6.081, p \ 0.05, one-way ANOVA followed by the post hoc Bonferroni test). These suggested exposure to IH induced anxiety in mice, which could be prevented by the antioxidant/NOX inhibitor apocynin administration and genetically gp91-phox/NOX2 KO in mice. All groups made a similar number of total-arms entries (Fig. 7c; F3,39 = 3.983, p = 0.874), suggesting that the reduced open arm activity in WT mice exposed to IH was due to increased anxiety, but not hypoactivity or motor impairment. The number of SAPs which is categorized as risk assessment and has been considered closed related to anxiety, was significantly increased in WT mice exposed to IH compared to SA, IH ? APO and IH ? KO mice (Fig. 7d; F3,39 = 7.259, p \ 0.05, one-way ANOVA followed by the post hoc Bonferroni test). These results suggest that exposure to IH has tendency to induce mental and psychiatric anxiety in mice and the NOX2 might mediate the IH-induced mental dysfunction.
Exposure to IH Induced Mental and Psychiatric Anxiety in Mice For a further study of psychiatric symptoms after IH exposures in WT mice and to investigate the relationship between
Fig. 6 Emotional responses and habituation to the novel environment were measured by an open-field test for 10 min. a Locomotion activity in the open field test among SA, IH, IH ? APO and IH ? KO groups. b The time spent in the center and c the number of
entries into the center are analyzed among SA, IH, IH ? APO and IH ? KO groups. (mean ± SEM; n = 10; *p B 0.05, **p B 0.01 vs. SA group; #p B 0.05, ##p B 0.01 vs. IH group)
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Fig. 7 Exposure to IH induces anxiety in mice. WT mice exposed to IH a spent significantly less time in the open arm and b reduced number of open-arm entries in the elevated plus maze compared to SA, IH ? APO and IH ? KO group. c All groups made a similar
number of total-arms entries. d The number of SAPs increased in WT mice exposed to IH compared to SA, IH ? APO and IH ? KO group. (mean ± SEM; n = 10; *p B 0.05, **p B 0.01 vs. SA group; # p B 0.05, ##p B 0.01 vs. IH group)
Exposure to IH Induced Deficits in Contextual and Cued Fear Conditioning
apocynin administration and genetically gp91-phox/NOX2 KO in mice (p \ 0.05). These suggest that exposure to IH induced long-term memory impairment in mice and the NOX2 might mediate the IH-induced cognitive deficit.
To assess the influence of exposure to IH on the long-term memory and investigate the relationship between NOX2dependent oxidative stress and cognitive impairment in mice, WT mice underwent contextual/cued fear conditioning test. In this paradigm, mice learn to associate previous neutral auditory cues and the apparatus (context) with electric foot shock in a single training session, such that robust long-term memory was established for an experimental context and an auditory cue. Long-term memory was assessed based on the freezing reaction of the mice in response to the context or the conditioned cue. The freezing response to the same context in WT mice exposed to IH was reduced significantly compared with SA group after a 48-h retention delay (Fig. 8a; F3,39 = 11.289, p = 0.026, one-way ANOVA followed by the post hoc Bonferroni test). The cued fear conditioning was also reduced significantly after a 48-h retention delay compared with SA mice (Fig. 8b; F3,39 = 13.486, p = 0.007, oneway ANOVA followed by the post hoc Bonferroni test). The results showed the cognitive impairment was significantly attenuated by the antioxidant/NOX inhibitor
Fig. 8 Mice from different groups were subjected to contextual/cued fear conditioning. a Freezing response was measured in the context before shock (basal freezing) and in the conditioning chamber (contextual fear response) 48 h after conditioning. b Freezing
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Discussion The relationship among IH-induced neuropathological alterations, HIF-1a, NOX2, and oxidative stress in the brain was determined in the present study. We demonstrated (Fig. 9) that WT mice exposed to IH led to an upregulation of NOX2 and p47-phox expression. The upregulation of NADPH-Oxidase activity was accompanied by signs of brain oxidative stress and loss of PV-IN in the frontal cortex. The behavioral alterations were also accompanied with these neuropathological alterations. The antioxidant/ NOX inhibitor apocynin and genetically gp91-phox/NOX2 KO in mice prevented signs of oxidative stress, the decrease of PV-IN in the brain, and the behavioral alterations induced by IH exposures. There is increasing evidence IH exposures lead to oxidative stress in the brain in rodent models [19]. Prolonged oxidative stress has been proposed to contribute to the
response (for the same set of mice) was measured in an alternative context without auditory cue (basal freezing) or with a cue after contextual text. (mean ± SEM; n = 10; *p B 0.05, **p B 0.01 vs. SA group; #p B 0.05 vs. IH group)
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Fig. 9 a We demonstrated IH exposures induced increase of HIF-1a in the prefrontal cortex, which then facilitated the increase of NOX2 expression and heightened the NOX2 activity which led to further ROS generation. The heightened NOX2 activity activated HIF-1a in a ROS-dependent manner and created a feed-forward mechanism that was essential for the pathogenesis of ROS generation in prefrontal cortex after IH exposures. The increased ROS accumulation ultimately contributed to PV-IN loss in brain and led to psychiatric diseases and behavioral alterations. b Experimental design of the study. WT or KO mice were subjected to IH or SA exposures for 14 days. WT mice exposed to IH were pretreated with either the antioxidant/NOX inhibitor apocynin or HIF-1a inhibitor digoxin, the neuropathological alterations and behavioral alterations was determined shortly after IH exposures
pathophysiology of mental disorders and contribute to deteriorating course and poor outcome in psychiatric patients [23]. Some persons with OSA have significant residual hypersomnolence and accompanied with long-term hypoxia/reoxygenation events. Long-term IH exposure in adult mice, simulating hypoxia/reoxygenation events in persons and patterns of moderate–severe sleep apnea, result in lasting hypersomnolence, oxidative injury, and proinflammatory responses in wake-active brain regions. ROS can be generated by various subcellular compartments, including mitochondria, the cellular membrane, lysosomes, peroxisomes, and the endoplasmic reticulum. Activation of NADPH oxidase leads to generation of the superoxide ion (O2-), a highly damaging ROS [24, 25]. To date, five NADPH oxidase enzyme (NOX) isoforms have been identified (NOX 1–5), and localization studies have shown that of the five NOX enzymes, the NOX2 and NOX4 isoforms are highly localized in cerebral cortex [26, 27]. In the present study, the NOX activity and oxidative stress in the prefrontal cortex of the NOX2 KO mice are not completely abolished, it is possible that other subunits or mechanisms may be of importance for the function of
NOX. Other homologues of the NOX, most importantly NOX4, might be also increased in models of intermittent hypoxia. It is thus possible that these enzymes provide an additional source of NOX activity and oxidative stress that is not affected by the genetic KO for the gp91-phox subunit. Our data suggest, however, that the NOX activity and oxidative stress were indeed significantly reduced in the NOX2 KO mice after exposed to IH, emphasizing the key role for NOX2 in the IH-induced oxidative stress. Our study provides insight into the functional role of NOX2 activation and its role in IH-induced oxidative stress. Previous studies [20]. Have found that long-term IH exposure activates brain NADPH oxidase and that this enzyme serves as a critical source of superoxide in the oxidation injury and in hypersomnolence. Excessive NADPH oxidase activity plays a role in IH-induced CNS dysfunction and neurobehavioral impairments [20]. The neuropsychological impairments are accompanied by increased levels of systemic markers of oxidative stress. HIF-1 is a heterodimeric protein composed of a constitutively expressed HIF-1b subunit and an O2-regulated HIF-1a subunit,which plays as a master regulator of O2 homeostasis that controls multiple physiological processes by regulating the expression of hundreds of genes [28, 29]. HIF-1a plays a pro-oxidant role in the setting of IH exposure by increasing NOX activity [30]. Previous studies [18] have reported that IH increased HIF-1a protein levels in the cerebral cortex of mice and demonstrated that (1) IH exposures activates HIF-1 in a ROSdependent manner and (2) HIF-1 is required for IH-induced ROS generation. In the present study, using multiple pharmacological and genetic approaches, we found IH-induced increase of NOX2 expression and NOX activity is mediated by HIF-1 in the CNS of mice. The heightened NOX2 activity activated HIF-1 in a ROS-dependent manner and created a feed-forward mechanism that was essential for the pathogenesis of ROS generation in the prefrontal cortex after IH. In neurons, NOX2 enzyme is thought to be involved in cell fate and modulation of neuronal activity [10]. From a pathologic point of view, NOX enzymes are involved in the increase of oxidative stress seen in a variety of brain disorders, from psychiatric to neurodegenerative diseases [11]. To confirm the role of NOX enzymes in the development of these brain pathologies, compounds that inhibit the activity of NOX proteins have been used in animal models [31]. Among the proposed NOX inhibitors, apocynin has been shown to have multiple biological actions [32]. Such as anti-inflammatory and antioxidant effects in cell and animal models. Even if apocynin cannot be considered a specific NOX inhibitor, the previous study have shown that it is effective at reducing NOX-dependent superoxide and oxidative stress in rodents [33].
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There is compelling epidemiologic evidence that IH exposure leads to oxidative stress and neuropathological alterations in the CNS. Long-term oxidative injury is involved in the development of mental and psychiatric diseases [34, 35]. Many hypotheses have been proposed on what mechanisms of pathogenesis process mediate these brain dysfunctions. Among them, loss of phenotype of PV expression in the brain has been proposed [15]. PV-IN in the brain are involved in the generation of gamma oscillations, which regulate working memory and information transmission between cortical areas [36, 37]. It has recently been suggested that the loss of PV expression and GABAergic function may be a central feature in the pathogenesis of schizophrenia [13, 16]. But whether these deficiencies were a consequence or a cause of the disorder in mice of this study was a matter of debate. Remarkable, decrease in PV-IN have been reported in the cerebral cortex in the perinatal rat anoxia models [38–40]. In the present study, we showed IH exposures led to an upregulation of NOX2 activity which was accompanied by signs of brain oxidative stress, decrease of PV immunoreactivity in neurons and behavioral alterations. The antioxidant/NOX inhibitor apocynin and genetically gp91phox/NOX2 KO in mice prevented signs of oxidative stress in the brain, the decrease of PV immunoreactivity, and the behavioral alterations. In conclusion (Fig. 9a), we demonstrated that the upregulation of NOX2 activity might be a central feature in the pathogenesis of IH-induced neuropathological alterations. Loss of PV expression are set to become interesting new players in the mechanisms leading to psychiatric diseases. NOX2-induced dysfunction of PV-IN might be a core feature for IH-induced behavioral alterations. Downregulation of NOX2 activity and oxidative stress might provide effective curative approaches for IH-induced behavioral alterations. The present study provide a molecular mechanism for the coupling between sleep apnea and neuropathological alterations, as well as potential new cellular mechanism for NOX2-induced neuropathological alterations. Acknowledgments This work was supported by the Grants 81172094 and 8110186 from the National Science Foundation of China, Grant 2011-WS-005 from the Six Talents Peak Foundation of Jiangsu Province. Conflict of interest We declare that we have no competing interests.
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ORIGINAL PAPER
Intermittent Hypoxia-Induced Parvalbumin-Immunoreactive Interneurons Loss and Neurobehavioral Impairment is Mediated by NADPH-Oxidase-2 Liang Yuan1 • Jing Wu1 • Jiang Liu2 • Guowei Li1 • Dong Liang3
Received: 23 December 2014 / Revised: 21 March 2015 / Accepted: 20 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Obstructive sleep apnea usually contribute to psychiatric diseases and cognitive impairments in adults. Loss of parvalbumin (PV)-immunoreactive interneurons (PV-IN) in the brain cortex is an important feature of psychiatric diseases, such as schizophrenia. Here we investigate the causal contribution of oxidative stress in the brain cortex to neuropathological alterations in a mouse model of sleep apnea. Wild-type (WT) and the NADPHoxidase-2 (gp91-phox/NOX2) knock-out adult male C57BL/6J mice were exposed to intermittent hypoxia (IH) or standard room air in the same chamber. In vivo we determined the impact (1) of IH exposures on NOX2 expression, (2) of genetic gp91-phox/NOX2 knock-out and (3) of pharmacological NOX2 inhibition on IH-induced neuropathological alterations in adult mice. Endpoints were oxidative stress, PV-IN and neurobehavioral alterations. The results showed IH exposures increased NOX2 expression in the prefrontal cortex of WT mice, which was accompanied with elevations of indirect markers of oxidative stress (HNE, HIF-1a, 8-OHDG). WT mice showed
Liang Yuan and Jing Wu have equally contributed to this work. & Dong Liang [email protected] 1
Department of Anaesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, People’s Republic of China
2
Comprehensive Cancer Center of Drum-Tower Hospital, School of Medicine, Nanjing University, Nanjing, People’s Republic of China
3
Department of Emergency, Ruijin Hospital North, School of Medicine, Shanghai Jiaotong University, 999 Xiwang Road, Jiading District, Shanghai 201801, People’s Republic of China
loss of PV-IN in the prefrontal cortex and increased locomotion activity and anxiety levels after exposed to IH, while no change emerged in NOX2 knock-out mice. Treatment of WT mice with the antioxidant/NOX inhibitor apocynin prevented the neuropathological and neurobehavioral alterations induced by IH exposures. Our data suggest that NOX2-derived oxidative stress is involved in the loss of PV-IN in the prefrontal cortex and development of neurobehavioral alterations for adult mice exposed to IH. These results provide a molecular mechanism for the coupling between sleep apnea and brain oxidative stress as well as potential new therapeutic avenues. Keywords Intermittent hypoxia Oxidative stress NADPH-oxidase-2 PV-immunoreactive interneurons Neurobehavioral alterations
Introduction Obstructive sleep apnea (OSA), a clinical syndrome that is characterized by repeated episodes of upper airway obstruction during sleep, is a significant and highly prevalent health problem, due not only to its cardiovascular and metabolic morbidity, but also because of the prominent cognitive and behavioral implications of the disease. The neuropsychological impairments are accompanied by increased levels of systemic markers of oxidative stress and inflammation in nervous system [1, 2]. The inordinate sensitivity of neuronal tissues to alterations in oxygen homeostasis has led to the hypothesis that the neuropathological and neurobehavioral alterations observed in OSA patients are associated, at least in part, with the episodic hypoxia-reoxygenation cycles during sleep that characterize OSA. In support of this hypothesis, animal
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models have demonstrated that chronic exposure to IH is accompanied with neurodegenerative changes, increased oxidative stress, and impaired spatial learning and memory in the morris water maze [3–6]. Genetic or pharmacological manipulations of oxidative stress pathways attenuated IH-induced deficits in neuropathological and neurobehavioral alterations. NADPH oxidase is composed of catalytic and regulatory subunits which include two membrane-bound subunits (gp91-phox and p22-phox) and three cytosolic subunits, which include p47-phox, p67-phox, and Rac [7]. The membrane-bound subunits form a heterodimer that stabilizes them within the membrane, whereas the cytosolic subunits are recruited to the membrane following stimulation. Complete complex assembly is necessary for full NADPH oxidase activity [8]. Neurons express the NOX2 isoform of NADPH oxidase, which contains the gp91-phox catalytic subunit and requires the p47-phox assembly subunit [9]. In neurons, NOX2 enzyme is thought to be involved in cell fate and modulation of neuronal activity [10]. From a pathologic point of view, NOX enzymes are involved in the increase of oxidative stress seen in a variety of brain disorders, from psychiatric to neurodegenerative diseases [11, 12]. Cortical interneurons are classified into several subtypes that contribute to cortical oscillatory activity. PV-expressing cells, a type of inhibitory interneuron, are involved in the gamma oscillations of local field potentials (LFPs). PV-expressing cells are the major type of inhibitory gammaaminobutyric acid-producing (GABAergic) interneurons of the neocortex. These cells are distributed across cortical layers, target the proximal regions of pyramidal cells, and are characterized by a fast-spiking physiological phenotype. Loss of phenotype of PV-IN in the brain cortex and psychiatric behavioral alterations are often reported in the previous studies [13, 14], which are regarded as the important features of psychiatric diseases, such as schizophrenia [15–17]. Exposure to IH, a hallmark of OSA, is associated with increased oxidative stress in the central nervous system (CNS) and neurobehavioral impairments. HIF-1a (hypoxia-inducible factor-1a) is an indirect marker for oxidative stress, excessive NADPH oxidase activity and HIF1a expression has been shown in IH-induced neuropathological alterations [18–20]. HIF-1a is required for IH-induced reactive oxygen species (ROS) generation and plays a pro-oxidant role in the setting of IH exposure [18]. In the present study, we proposed loss of PV-IN in the brain was an important feature of IH-induced neuropathological alterations. By using multiple pharmacological and genetic approaches, we evaluate the possible role of oxidative stress in the development of neuropathological and neurobehavioral alterations induced by IH.
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Materials and Methods Animals NADPH-oxidase-2 knock-out (KO) C57BL/6J mice were originally obtained from Jackson Laboratories and were bred with wild-type (WT) C57BL/6J mice at the Animal Center of Jinling Hospital, Nanjing, China, with free access to food and water. Littermate KO and WT animals were bred from heterozygote (±) females and KO (0/-) males resulting in WT (0/?) and KO (0/-) male littermates. Genotyping of animals used in the experiment was performed as previously described [8]. The primers (Cyber-gene) showed either the WT (50 -AAGAGAAACTCCTCTGCTGTGAA-30 ) with a band of 240 bp or the KO (5-GTTCTAATTCCATCAG AAGCTTATCG-30 ) with a band of 195 bp in addition to the common band (50 -CGCACT GGAACCCCTGAGAAA GG-30 ). The PCR reaction consisted of 12 cycles: denaturation: 94 °C, 20 s; annealing: 64 °C, 30 s; elongation: 72 °C, 35 s followed by an additional 25 cycles: denaturation: 94 °C, 20 s; annealing: 58 °C, 30 s; elongation: 72 °C, 35 s. The PCR products were analyzed by using TBE agarose electrophoresis (1.5 %), labeled with ethidium bromide. The animal experiments were approved by the Ethics Committee of Jin-ling Hospital, Nanjing University.
Experimental Design of the Study The experimental design were as follows (see Fig. 9b): in the first set of experiments: the authors determined the impact of IH on NOX2 expression and neuropathological alterations in the WT mice. The apocynin was used to determine the effect of pharmacological NOX2 inhibition on IH-induced neuropathological alterations. For the experiment, WT mice were treated either with apocynin (APO, Sigma, St. Louis, MO, USA; 4 mg/kg) or vehicle (VEH: 0.5 % DMSO) by intraperitoneal (i.p.) injection 30 min before and immediately after the exposure to 8 h daily for 14 days of IH. In the second set of experiments: the authors determined the effect of genetically NOX2 KO in mice on IH-induced neuropathological alterations. In the third set of experiments: the authors determined the effect of digoxin (D, HIF-1a inhibitor) on IH-induced NOX2 expression in the WT mice and investigated whether IH-induced expression of NOX2 was mediated by HIF-1a. For the experiment, WT mice were treated either with digoxin (Sigma, St. Louis, MO, USA; 1 mg/ kg, i.p.) or vehicle (VEH: 0.5 % DMSO, i.p.) 30 min before and immediately after the exposure to 8 h daily for 14 days of IH exposures.
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Intermittent Hypoxia Exposures WT (0/?) and KO (0/-) male (body weight, 20–25 g) housed in feeding cages were exposed to IH for 8 h per day for 14 days as previously described (IH group) [18]. Briefly, mice were placed in a specialized chamber, which was flushed with alternating cycles of pure nitrogen and compressed standard room air (SA). The IH profile consisted of alternating SA and nitrogen every 90 s. During hypoxia, inspired O2 levels rapidly reached a nadir of 5 % O2. Oxygen concentration was continuously measured by an O2 analyzer, and was changed by a computerized system controlling gas outlets. Control experiments were exposed to alternating cycles flushing SA every 90 s instead of nitrogen. Behavioral Testing For study of IH-induced neuropathological and psychiatric alterations, behavioral alterations in open-field test, elevated plus maze (EPM), forced swimming test (FST), fear conditioning test were carried out consecutively after IH exposure. The movement of each mouse was monitored and analyzed using a computer-operated video tracking system. The apparatus was cleaned after each trial. All apparatus used in tests were come from Shanghai Soft maze Information Technology Co, Ltd, China. All mice after behavioral tests were not used for any biochemistry studies and would be administered euthanasia. Open Field Emotional responses to the novel environment were measured by an open-field test. The open field task approaches the conflict between the innate fear that rodents have of the central area of a novel or brightly lit open field versus their desire to explore new environments. When anxious, the natural tendency of rodents is to prefer staying close to the walls (thigmotaxis). In this context, anxiety-related behavior is measured by the degree to which mice avoid the center of the open field test. Mice were placed in an openfield (40 9 40 9 40 cm) chamber made of white acrylic for 10 min under 70 lux lighting conditions, in which the video tracking system quantifies the number of entries into and time spent in the center of the locomotion arena. Activity was measured as the total distance traveled. Elevated Plus Maze (EPM) The EPM was used to assess anxiety. The basic measure is the animal preference for dark, enclosed places over bright, exposed places. Mice prefer to enter into closed arms compared to open arms. Time spent in the dark area is viewed as avoidance or anxiety-like behavior. Mice were
placed in the center of the maze facing a closed arm, and allowed to explore for 10 min in isolation. The following parameters were scored: (a) percent time spent in open arms and number of entries to open arms; (b) total number of entries to arms; (c) the number of stretched-attend postures (SAPs), defined as a posture in which the subject stretches forward and then retracts to its original position, performed from the central platform or enclosed-arms towards open-arms, was also recorded. This latter response is categorized as risk assessment, and has also been considered closed related to anxiety [21]. Fear Conditioning Fear conditioning was performed in a black plastic chamber (XR-XC404) equipped with a stainless steel grid floor. The conditioning (acquisition) trial for contextual and cued fear conditioning consisted of a 2 min exploration period followed by three conditioned stimulus (CS)–unconditioned stimulus (US) pairings separated by 1 min each (US, 0.70 mA footshock intensity, 2 s duration; CS, 70 db white noise, 20 s duration; US was delivered during the last 2 s of the CS presentation). A contextual test was performed in the conditioning chamber for 3 min in the absence of white noise at 48 h after conditioning. A cued test (for the same set of mice) was performed by presentation of the cue (70 db white noise, 3 min duration) in an alternative context with distinct visual and tactile cues. The cued test was conducted after the contextual test was finished. The rate of freezing response of mice was used to measure the fear memory. The level of nonspecific freezing provoked by the new context was controlled for 2 min before the presentation of the cue in that new context. Brain Tissue Harvest and Protein Level Quantification Different groups of mice which have not been subjected to behavior tests were used for biochemistry studies after IH exposure. In brief, the mice were anesthetized by sodium pentobarbital (50 mg/kg) followed by transcardial perfusion with paraformaldehyde 4 % in phosphate buffered saline (PBS; pH 7.35) for immunohistochemistry studies or PBS for western blot studies. For the western blot analysis, the harvested brain tissues were homogenized on ice using immunoprecipitation buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, and 0.5 % Nonidet P-40) plus protease inhibitors (1 lg/ml aprotinin, 1 lg/ml leupeptin, and 1 lg/ml pepstatin A) as described in previous studies. The lysates were collected, centrifuged at 12,000 rpm for 10 min, and quantified for total protein with the bicinchoninic acid protein assay kit (Pierce Technology Co., Iselin, NJ, USA).
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Western Blot Analysis The samples were subjected to western blot analysis according to the standard procedure using antibodies for hypoxia-inducible factor 1a (HIF-1a) (1:500, #sc-10790, Santa Cruz Biotechnology, Santa Cruz, CA, USA), NOX2 (1:2000, #ab129068, Abcam, Cambridge, MA, USA), p47phox (1:1000, #sc-7660, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and b-actin (1:10,000, Sigma, St. Louis, MO, USA). Quantitation of bands was undertaken using the Image J software (NIH Image, USA). We quantified the western blots bands by using b-actin levels to normalize protein levels (e.g., determining the ratio of NOX2 to b-actin amount) and control for loading differences in the total protein amount. Immunohistochemistry PV-IN in the prefrontal cortex were evaluated by immunohistochemical staining after IH exposure. The immunohistochemistry was performed according to the standard procedure. Every six sections were used for staining. The sections were deparaffinized, washed and incubated with PV antibody (1:1000; #mab1572; Millipore) and biotinylated secondary antibody. The PV-positive cells in the mouse cortex were counted manually in five randomly selected areas under a 20 9 objective microscope by an investigator who was blinded to the experiments. Six brains from each group were used for immunohistochemistry alalysis, and six brain sections of 5 lm thickness were examined in each brain. Data are given as mean number of cells/mm2. Measurement of NADPH Oxidase Activity The prefrontal cortex tissues were isolated and homogenized in a RIPA buffer. The homogenate was subjected to centrifugation at 250g for 10 min to remove cellular debris. Supernatant was then centrifuged at 20,000g for 20 min at 4 °C to eliminate mitochondria, lysosomes, peroxisomes, Golgi membranes, and rough endoplasmic reticulum. The resulting supernatant was centrifuged at 100,000g for 60 min at 4 °C. The pelleted plasma membrane fraction containing NOX was dissolved in a buffer containing 8 mM piperazine-N, N’-bis 2-ethanesulfonic acid (pH 7.2), 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, and proteolytic inhibitors. The solubilized membrane fraction samples were placed in a multiwell plate. NOX activity was measured using a cytochrome c reduction assay [18]. Briefly, 100 lg aliquots of membrane proteins were incubated in 25 mM HEPES buffer (pH 7.0) with 150 lm cytochrome c, 100 lm NADPH for 30 min at 37 °C in the presence and
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absence of superoxide dismutase (SOD; 200 U/ml). Cytochrome c reduction was measured by determining absorbance at 550 nm. The amount of SOD-inhibitable NOX activity expressed in nmol/min/mg protein was calculated using an extinction coefficient of 21 mM-1cm-1. Measurement of Lipid Peroxidation in the Prefrontal Cortex Since the most abundant lipid peroxidation product is 4-hydroxy-2-nonenal (HNE), measurement of this product has been used as an indicator of lipid peroxidation. The samples were subjected to analysis as previously described [22]. The prefrontal cortex was used for measurement of HNE using the bioxytech HAE-586 spectrophotometric assay kit (OxisResearch, Portland, OR, USA). The HNE concentrations in experimental samples were determined against HNE standards provided in the assay kit. Measurement of 8-Hydroxydeoxyguanosine (8-OHDG) The mice were anesthetized by sodium pentobarbital (50 mg/ kg) followed by transcardial perfusion with phosphate buffered saline (PBS; pH 7.35) for 5 min and the prefrontal cortex was dissected, snap frozen in liquid nitrogen, and stored at –80 °C until assay the following day. Cortical tissues were homogenized in 20 mM phosphate buffer (pH 7.4) containing 0.5 mM butylated hydroxytoluene to prevent sample oxidation. Levels of 8-OHDG (an oxidized form of deoxyguanosine that is a widely used biomarker of in vivo oxidative DNA damage) were measured in the prefrontal cortex using a commercially available assay (Cell Biolabs, San Diego, CA, USA) [20]. Briefly, cortical samples or 8-OHDG standards were first added to an 8-OHDG/BSA conjugate preabsorbed enzyme immunoassay plate. After a brief incubation, an anti—8-OHDG mAb was added, followed by a horseradish peroxidase-conjugated secondary antibody. The 8-OHDG content in the cortical samples was then determined by comparison with the 8-OHDG standard curve. Statistical Analysis Data regarding biochemistry changes and behavioral tests were expressed as mean ± SEM and analyzed by the Statistical Product for Social Sciences (SPSS; version 16.0, IL, USA). Normal distribution of data was analyzed using the Kolmogorov–Smirnov test. The data were performed using the student two-sample t test or one-way ANOVA followed by the post hoc Bonferroni test. When data did not fulfill parametric assumptions, data were expressed as medians and interquartile ranges and were analyzed using nonparametric tests (Kruskall–Wallis test with Dunn’s
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multiple comparisons test). In all experiments, differences were considered statistically significant at p B 0.05.
NOX-related oxidative stress in the loss of PV immunoreactivity induced by IH exposures.
Results
IH Increased HIF-a Expression in the Prefrontal Cortex
IH Exposures Induced Loss of PV-IN in the Prefrontal Cortex The authors determined alterations of the number of PV-IN in the prefrontal cortex to study the effect of IH exposures on brain neuropathological alterations and investigated the relationship between NOX2-dependent oxidative stress and the decrease in PV immunoreactivity. The results showed that IH exposures in mice induced loss of PV-IN in the prefrontal cortex compared with SA, which was attenuated by pre-treatment with apocynin before IH exposures or in gp91-phox/NOX2 KO mice (Fig. 1; F3,23 = 14.763, p \ 0.05, one-way ANOVA followed by the post hoc Bonferroni test). These findings suggest a possible role of
In accordance with the previous studies [18], we showed IH increased HIF-a expression in the prefrontal cortex (Fig. 2; p = 0.002, Kruskall–Wallis test with Dunn’s multiple comparisons test). In the present study, using multiple pharmacological and genetic approaches, the increased HIF-a expression was attenuated in gp91-phox/ NOX2 KO mice or by pretreatment with either the antioxidant/NOX inhibitor apocynin or HIF-1a inhibitor digoxin (p \ 0.05, Kruskall–Wallis test with Dunn’s multiple comparisons test), suggesting IH activated HIF-1a in a NOX2-dependent manner. HIF-1a Mediated the Increase of NOX2 Expression and Activity in Response to IH The results (Figs. 3a, 4) demonstrated IH exposures led to a substantial increase in NOX2 expression and activity in the prefrontal cortex in WT mice (p \ 0.01, Kruskall–Wallis test with Dunn’s multiple comparisons test). Such IH-induced increase of NOX2 expression and activity were much attenuated in gp91-phox/NOX2 KO mice and by pretreatment of antioxidant/NOX inhibitor apocynin
Fig. 1 IH exposures induced loss of PV-IN in the prefrontal cortex, which was attenuated in gp91-phox/NOX2 knock-out mice or pretreatment with apocynin before IH exposures. a Representative images of PV-IN in the prefrontal cortex. (original magnification 9200). b Quantification of the immunohistochemistry image. Data are given as mean number of cells/mm2. (mean ± SEM; n = 6; **p B 0.01 versus SA group; #p B 0.05 versus IH group)
Fig. 2 IH exposures induced HIF-1a expression in the prefrontal cortex of WT mice, which was prevented by HIF-1a inhibitor digoxin and the antioxidant/NOX inhibitor apocynin. Gp91-phox/NOX2 knock-out in mice also prevented the IH-induced HIF-1a expression in the prefrontal cortex of mice. a Representative western blot images of HIF-1a protein in the prefrontal cortex of all groups of mice to illustrate band intensities. b Histogram showed results of the densitometric analysis of western blot images of HIF-1a. (mean ± SEM; n = 6; **p B 0.01 vs. SA group; #p B 0.05, ##p B 0.01 vs. IH group)
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Fig. 3 IH exposures induced NOX2 expression in the prefrontal cortex of WT mice which was prevented by HIF-1a inhibitor digoxin. Gp91-phox/NOX2 knock-out in mice completely prevented the IHinduced NOX2 expression in the prefrontal cortex of mice. a Western blot showed HIF-1a inhibitor digoxin prevented the IH-induced NOX2 expression in the prefrontal cortex of WT mice. Lack of the
NOX2 band in the brain proteins extracted from the gp91-phox/ NOX2 knock-out mice (far right lane) confirmed specificity of the anti-NOX2 antibody and location of NOX2 specific band. b Western showed upregulation of the cytosolic subunits p47-phox after IH exposures. (mean ± SEM; n = 6; **p \ 0.01, ***p B 0.001 vs. SA group; ##p B 0.01 vs. IH group)
Indirect Markers of Oxidative Stress (HNE, 8-OHDG) were Increased After IH Exposures in the Prefrontal Cortex of WT Mice
Fig. 4 NADPH oxidase activity in the prefrontal cortex was measured as NADPH-dependent cytochrome c reduction. IH exposures resulted in a substantial increase in NOX activity in WT mice. Such IH-induced increase of NADPH oxidase activity were much attenuated in gp91-phox/NOX2 knock-out mice and by pretreatment of HIF-1a inhibitor digoxin and antioxidant/NOX inhibitor apocynin. (mean ± SEM; n = 6; **p B 0.01 vs. SA group; #p B 0.05, ## p B 0.01 vs. IH group)
(p \ 0.05, Kruskall–Wallis test with Dunn’s multiple comparisons test). To investigate whether HIF-1 is required for IH-induced NOX2 expression and activity, a pharmacological approach targeting inhibiting HIF-1a protein synthesis [18] was employed. The results showed the IHincreased NOX2 expression and activity in the prefrontal cortex of WT mice was prevented by HIF-1a inhibitor digoxin (p \ 0.05, Kruskall–Wallis test with Dunn’s multiple comparisons test). Lack of the NOX2 band in brain proteins extracted from a gp91-phox/NOX2 KO mice confirmed specificity of the anti-Nox2 antibody (Fig. 3a). IH exposures upregulated the cytosolic subunits p47-phox which was necessary for full NADPH oxidase activity (Fig. 3b; p = 0.007, Student’s t test).
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In the present study, the authors showed IH exposures induced indirect markers of oxidative stress (HNE, 8-OHDG) in the prefrontal contex of WT mice (HNE: Fig. 5a; p = 0.002, Kruskall–Wallis test with Dunn’s multiple comparisons test; 8-OHDG: Fig. 5b; F4,29 = 13.775, p = 0.006, one-way ANOVA followed by the post hoc Bonferroni test). To test whether the signs of oxidative stress detected in the brain of IH-exposed mice could be prevented pharmacologically or genetically, the antioxidant/NOX inhibitor apocynin, HIF-1a inhibitor digoxin and genetically gp91phox/NOX2 KO in mice was employed. Results showed that the administration of digoxin, apocynin and genetically gp91-phox/NOX2 KO in mice prevented IH-induced lipid peroxidation (Fig. 5a) and DNA oxidation (Fig. 5b) in the prefrontal contex after IH exposures (p \ 0.05). IH Exposures Increased the Spontaneous Locomotion Activity and Anxiety-Related Behavior in Mice To correlate molecular changes with behavioral observations and investigate the relationship between NOX2-dependent oxidative stress and behavioral alterations in mice, emotional responses and habituation to the novel environment were measured by an open-field test for 10 min. Open field is a sensorimotor test for locomotion, habituation, exploratory, and emotional behavior, including risk assessment and anxiety-like behavior, in novel
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Fig. 5 IH exposures induced lipid peroxidation and DNA oxidation in the prefrontal contex. The administration of HIF-1a inhibitor digoxin, antioxidant/NOX inhibitor apocynin and gp91-phox/NOX2
knock-out in mice prevented IH-induced a lipid peroxidation and b DNA oxidation in the prefrontal contex. (mean ± SEM; n = 6; **p B 0.01 vs. SA group; #p B 0.05, ##p B 0.01 vs. IH group)
environments. The open field was performed with a focus on behavioral functions that are physiologically regulated by the prefrontal cortex, such as spontaneous locomotion activity. Behavioral tests showed an increase in spontaneous locomotion activity (Fig. 6a; F3,36 = 7.681, p = 0.006, one-way ANOVA followed by the post hoc Bonferroni test) and a decrease of time in centre and number of center entries in IH-exposed mice compared with SA-exposed mice (Fig. 6b; F3,39 = 9.425, p = 0.027; Fig. 6c; F3,39 = 11.594, p = 0.031; one-way ANOVA followed by the post hoc Bonferroni test). The antioxidant/ NOX inhibitor apocynin administration and genetically gp91-phox/NOX2 KO in mice attenuated the IH-induced increase in spontaneous locomotion activity (Fig. 6a; p \ 0.05) and decrease of time in centre (Fig. 6b; p \ 0.05) and number of center entries (Fig. 6c; p \ 0.05). The results suggested IH exposures increased the spontaneous locomotion activity and anxiety-related behavior in mice and might be associated with further development of psychiatric diseases.
NOX2-dependent oxidative stress and anxiety in mice, we performed the EPM test which is used to assess anxiety. The authors showed WT mice exposed to IH spent significantly less time in the open arm of the EPM compared to SA, IH ? APO and IH ? KO mice (Fig. 7a; F3,39 = 4.885, p \ 0.05, one-way ANOVA followed by the post hoc Bonferroni test). The number of entries into the open-arms was also significantly decreased in IH-exposed WT mice (Fig. 7b; F3,39 = 6.081, p \ 0.05, one-way ANOVA followed by the post hoc Bonferroni test). These suggested exposure to IH induced anxiety in mice, which could be prevented by the antioxidant/NOX inhibitor apocynin administration and genetically gp91-phox/NOX2 KO in mice. All groups made a similar number of total-arms entries (Fig. 7c; F3,39 = 3.983, p = 0.874), suggesting that the reduced open arm activity in WT mice exposed to IH was due to increased anxiety, but not hypoactivity or motor impairment. The number of SAPs which is categorized as risk assessment and has been considered closed related to anxiety, was significantly increased in WT mice exposed to IH compared to SA, IH ? APO and IH ? KO mice (Fig. 7d; F3,39 = 7.259, p \ 0.05, one-way ANOVA followed by the post hoc Bonferroni test). These results suggest that exposure to IH has tendency to induce mental and psychiatric anxiety in mice and the NOX2 might mediate the IH-induced mental dysfunction.
Exposure to IH Induced Mental and Psychiatric Anxiety in Mice For a further study of psychiatric symptoms after IH exposures in WT mice and to investigate the relationship between
Fig. 6 Emotional responses and habituation to the novel environment were measured by an open-field test for 10 min. a Locomotion activity in the open field test among SA, IH, IH ? APO and IH ? KO groups. b The time spent in the center and c the number of
entries into the center are analyzed among SA, IH, IH ? APO and IH ? KO groups. (mean ± SEM; n = 10; *p B 0.05, **p B 0.01 vs. SA group; #p B 0.05, ##p B 0.01 vs. IH group)
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Fig. 7 Exposure to IH induces anxiety in mice. WT mice exposed to IH a spent significantly less time in the open arm and b reduced number of open-arm entries in the elevated plus maze compared to SA, IH ? APO and IH ? KO group. c All groups made a similar
number of total-arms entries. d The number of SAPs increased in WT mice exposed to IH compared to SA, IH ? APO and IH ? KO group. (mean ± SEM; n = 10; *p B 0.05, **p B 0.01 vs. SA group; # p B 0.05, ##p B 0.01 vs. IH group)
Exposure to IH Induced Deficits in Contextual and Cued Fear Conditioning
apocynin administration and genetically gp91-phox/NOX2 KO in mice (p \ 0.05). These suggest that exposure to IH induced long-term memory impairment in mice and the NOX2 might mediate the IH-induced cognitive deficit.
To assess the influence of exposure to IH on the long-term memory and investigate the relationship between NOX2dependent oxidative stress and cognitive impairment in mice, WT mice underwent contextual/cued fear conditioning test. In this paradigm, mice learn to associate previous neutral auditory cues and the apparatus (context) with electric foot shock in a single training session, such that robust long-term memory was established for an experimental context and an auditory cue. Long-term memory was assessed based on the freezing reaction of the mice in response to the context or the conditioned cue. The freezing response to the same context in WT mice exposed to IH was reduced significantly compared with SA group after a 48-h retention delay (Fig. 8a; F3,39 = 11.289, p = 0.026, one-way ANOVA followed by the post hoc Bonferroni test). The cued fear conditioning was also reduced significantly after a 48-h retention delay compared with SA mice (Fig. 8b; F3,39 = 13.486, p = 0.007, oneway ANOVA followed by the post hoc Bonferroni test). The results showed the cognitive impairment was significantly attenuated by the antioxidant/NOX inhibitor
Fig. 8 Mice from different groups were subjected to contextual/cued fear conditioning. a Freezing response was measured in the context before shock (basal freezing) and in the conditioning chamber (contextual fear response) 48 h after conditioning. b Freezing
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Discussion The relationship among IH-induced neuropathological alterations, HIF-1a, NOX2, and oxidative stress in the brain was determined in the present study. We demonstrated (Fig. 9) that WT mice exposed to IH led to an upregulation of NOX2 and p47-phox expression. The upregulation of NADPH-Oxidase activity was accompanied by signs of brain oxidative stress and loss of PV-IN in the frontal cortex. The behavioral alterations were also accompanied with these neuropathological alterations. The antioxidant/ NOX inhibitor apocynin and genetically gp91-phox/NOX2 KO in mice prevented signs of oxidative stress, the decrease of PV-IN in the brain, and the behavioral alterations induced by IH exposures. There is increasing evidence IH exposures lead to oxidative stress in the brain in rodent models [19]. Prolonged oxidative stress has been proposed to contribute to the
response (for the same set of mice) was measured in an alternative context without auditory cue (basal freezing) or with a cue after contextual text. (mean ± SEM; n = 10; *p B 0.05, **p B 0.01 vs. SA group; #p B 0.05 vs. IH group)
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Fig. 9 a We demonstrated IH exposures induced increase of HIF-1a in the prefrontal cortex, which then facilitated the increase of NOX2 expression and heightened the NOX2 activity which led to further ROS generation. The heightened NOX2 activity activated HIF-1a in a ROS-dependent manner and created a feed-forward mechanism that was essential for the pathogenesis of ROS generation in prefrontal cortex after IH exposures. The increased ROS accumulation ultimately contributed to PV-IN loss in brain and led to psychiatric diseases and behavioral alterations. b Experimental design of the study. WT or KO mice were subjected to IH or SA exposures for 14 days. WT mice exposed to IH were pretreated with either the antioxidant/NOX inhibitor apocynin or HIF-1a inhibitor digoxin, the neuropathological alterations and behavioral alterations was determined shortly after IH exposures
pathophysiology of mental disorders and contribute to deteriorating course and poor outcome in psychiatric patients [23]. Some persons with OSA have significant residual hypersomnolence and accompanied with long-term hypoxia/reoxygenation events. Long-term IH exposure in adult mice, simulating hypoxia/reoxygenation events in persons and patterns of moderate–severe sleep apnea, result in lasting hypersomnolence, oxidative injury, and proinflammatory responses in wake-active brain regions. ROS can be generated by various subcellular compartments, including mitochondria, the cellular membrane, lysosomes, peroxisomes, and the endoplasmic reticulum. Activation of NADPH oxidase leads to generation of the superoxide ion (O2-), a highly damaging ROS [24, 25]. To date, five NADPH oxidase enzyme (NOX) isoforms have been identified (NOX 1–5), and localization studies have shown that of the five NOX enzymes, the NOX2 and NOX4 isoforms are highly localized in cerebral cortex [26, 27]. In the present study, the NOX activity and oxidative stress in the prefrontal cortex of the NOX2 KO mice are not completely abolished, it is possible that other subunits or mechanisms may be of importance for the function of
NOX. Other homologues of the NOX, most importantly NOX4, might be also increased in models of intermittent hypoxia. It is thus possible that these enzymes provide an additional source of NOX activity and oxidative stress that is not affected by the genetic KO for the gp91-phox subunit. Our data suggest, however, that the NOX activity and oxidative stress were indeed significantly reduced in the NOX2 KO mice after exposed to IH, emphasizing the key role for NOX2 in the IH-induced oxidative stress. Our study provides insight into the functional role of NOX2 activation and its role in IH-induced oxidative stress. Previous studies [20]. Have found that long-term IH exposure activates brain NADPH oxidase and that this enzyme serves as a critical source of superoxide in the oxidation injury and in hypersomnolence. Excessive NADPH oxidase activity plays a role in IH-induced CNS dysfunction and neurobehavioral impairments [20]. The neuropsychological impairments are accompanied by increased levels of systemic markers of oxidative stress. HIF-1 is a heterodimeric protein composed of a constitutively expressed HIF-1b subunit and an O2-regulated HIF-1a subunit,which plays as a master regulator of O2 homeostasis that controls multiple physiological processes by regulating the expression of hundreds of genes [28, 29]. HIF-1a plays a pro-oxidant role in the setting of IH exposure by increasing NOX activity [30]. Previous studies [18] have reported that IH increased HIF-1a protein levels in the cerebral cortex of mice and demonstrated that (1) IH exposures activates HIF-1 in a ROSdependent manner and (2) HIF-1 is required for IH-induced ROS generation. In the present study, using multiple pharmacological and genetic approaches, we found IH-induced increase of NOX2 expression and NOX activity is mediated by HIF-1 in the CNS of mice. The heightened NOX2 activity activated HIF-1 in a ROS-dependent manner and created a feed-forward mechanism that was essential for the pathogenesis of ROS generation in the prefrontal cortex after IH. In neurons, NOX2 enzyme is thought to be involved in cell fate and modulation of neuronal activity [10]. From a pathologic point of view, NOX enzymes are involved in the increase of oxidative stress seen in a variety of brain disorders, from psychiatric to neurodegenerative diseases [11]. To confirm the role of NOX enzymes in the development of these brain pathologies, compounds that inhibit the activity of NOX proteins have been used in animal models [31]. Among the proposed NOX inhibitors, apocynin has been shown to have multiple biological actions [32]. Such as anti-inflammatory and antioxidant effects in cell and animal models. Even if apocynin cannot be considered a specific NOX inhibitor, the previous study have shown that it is effective at reducing NOX-dependent superoxide and oxidative stress in rodents [33].
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There is compelling epidemiologic evidence that IH exposure leads to oxidative stress and neuropathological alterations in the CNS. Long-term oxidative injury is involved in the development of mental and psychiatric diseases [34, 35]. Many hypotheses have been proposed on what mechanisms of pathogenesis process mediate these brain dysfunctions. Among them, loss of phenotype of PV expression in the brain has been proposed [15]. PV-IN in the brain are involved in the generation of gamma oscillations, which regulate working memory and information transmission between cortical areas [36, 37]. It has recently been suggested that the loss of PV expression and GABAergic function may be a central feature in the pathogenesis of schizophrenia [13, 16]. But whether these deficiencies were a consequence or a cause of the disorder in mice of this study was a matter of debate. Remarkable, decrease in PV-IN have been reported in the cerebral cortex in the perinatal rat anoxia models [38–40]. In the present study, we showed IH exposures led to an upregulation of NOX2 activity which was accompanied by signs of brain oxidative stress, decrease of PV immunoreactivity in neurons and behavioral alterations. The antioxidant/NOX inhibitor apocynin and genetically gp91phox/NOX2 KO in mice prevented signs of oxidative stress in the brain, the decrease of PV immunoreactivity, and the behavioral alterations. In conclusion (Fig. 9a), we demonstrated that the upregulation of NOX2 activity might be a central feature in the pathogenesis of IH-induced neuropathological alterations. Loss of PV expression are set to become interesting new players in the mechanisms leading to psychiatric diseases. NOX2-induced dysfunction of PV-IN might be a core feature for IH-induced behavioral alterations. Downregulation of NOX2 activity and oxidative stress might provide effective curative approaches for IH-induced behavioral alterations. The present study provide a molecular mechanism for the coupling between sleep apnea and neuropathological alterations, as well as potential new cellular mechanism for NOX2-induced neuropathological alterations. Acknowledgments This work was supported by the Grants 81172094 and 8110186 from the National Science Foundation of China, Grant 2011-WS-005 from the Six Talents Peak Foundation of Jiangsu Province. Conflict of interest We declare that we have no competing interests.
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