Male C57BL/6J mice (20–22 grams) were purchased from Jackson Laboratories (Bar Harbor, Maine), housed in a 12 hr light/dark cycle (lights on from 7:00 am to 7:00 pm) at a constant temperature (26 ±1°C). Mice were housed in groups of four in standard clear polycarbonate cages, and were allowed access to food and water ad libitum. All behavioral experiments were performed during the light period (between 9:00 am and 12:30 pm). Mice were randomly assigned to either IH, SH, or room air (RA) exposures. The experimental protocols were approved by the Institutional Animal Use and Care Committee and are in close agreement with the Guide in the Care and Use of Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.
In a subset of mice, treatment with recombinant human erythropoietin (rhEPO; Roche, Mannheim, Germany) was carried out for the duration of IH exposures and until all behavioral testing was completed. rhEPO was dissolved in 0.1 mol/L phosphate-buffered saline (PBS) containing 0.1% mouse serum albumin (Sigma) at a stock concentration of 2500 IU/mL. EPO-vehicle consisted of PBS containing 0.1% mouse serum albumin. rhEPO was delivered by intraperitoneal (IP) injection at a dose of 5000 IU/kg body weight in a daily fashion. EPO-vehicle was delivered by IP injection with a volume corresponding to that of rhEPO injection. This dosage has been shown to effectively cross the blood brain barrier in rodents [22–24].
Intermittent and sustained hypoxia exposures
Animals were maintained in 4 identical commercially-designed chambers (30"x20"x20"; Oxycycler model A44XO, BioSpherix, Redfield, NY) operated under a 12 hour light–dark cycle (7:00 am-7:00 pm) for 14 days prior to behavioral testing. Oxygen concentration was continuously measured by an O2 analyzer, and was changed by a computerized system controlling gas outlets, as previously described , such as to generate stable initial oxyhemoglobin nadir values (SaO2) in the 65–72% range for SH, and alternating every 180 seconds with normoxia (SaO2 > 95%) for IH conditions. In addition, time-matched normoxic exposures (RA) were conducted. Ambient temperature was kept at 22–24°C.
The Morris water maze was used to assess spatial reference learning and memory, as well as working memory. The maze protocol is similar to that described by Morris  with modifications for mice. The maze consisted of a white circular pool, 1.4m in diameter and 0.6m in height, filled to a level of 35cm with water maintained at a temperature of 21°C (Morris 1984). Pool water was made opaque by addition of 150 ml of non-toxic white tempera paint. A Plexiglas escape platform (10 cm in diameter) was positioned 1 cm below the water surface and placed at various locations throughout the pool. Extramaze cues surrounding the maze were located at fixed locations, and visible to the mice while in the maze. Maze performance was recorded by a video camera suspended above the maze and interfaced with a video tracking system (HVS Imaging, Hampton, UK).
Briefly, a standard place-training reference memory task was initiated and conducted for 6 days on mice in the water maze following exposures to 14 days of IH, SH, or RA. One day prior to place learning, mice were habituated to the water maze during a free swim. Place learning was then assessed over six consecutive days using a spaced training regimen that has been demonstrated to produce optimal learning in mice . Each training session consisted of three trials separated by a 10 minute inter-trial interval (ITI). On a given daily session, each mouse was placed into the pool from 1 of 4 quasirandom start points (N, S, E or W) and allowed a maximum of 90 seconds to escape to the platform where the mice were allowed to stay for 15 sec. Mice that failed to escape were led to the platform. The position of the platform remained constant during the trials. 24 h following the final training session, the platform was removed for a probe trial to obtain measures of spatial bias. To assess the performance in the water maze, mean escape latencies and swim distance were analyzed.
Retention tests were carried out 14 days after acquisition of the task. In the retention test, performance in a single session (two trials) was assessed, and the mean average performance of the two trials was calculated.
Elevated plus maze (EPM)
The elevated plus maze (EPM) was used to assess anxiety. The basic measure is the animal preference for dark, enclosed places over bright, exposed places [27, 28]. A 60 w light was placed above the apparatus and the test was video taped by an overhead camera. Mice were placed in the center of the maze facing a closed arm, and allowed to explore for 10 min in isolation. Each mouse received one videotaped trial. 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. The following parameters were scored: (a) Percent time spent in open and closed arms; (b) number of entries to closed arms; (c) Time spent in the center. An arm entry was defined as the entry of all four feet into either one of the closed arm. Of note, the maze was cleaned with 30% ethanol between trials to remove any odor cues.
Forced swimming test (FST)
Briefly, mice were individually forced to swim in an open cylindrical container (diameter 14 cm, height 20 cm), with a depth of 15 cm of water at 25 ± 1°C. The immobility time, defined as the absence of escape-oriented behaviors, was scored during 6 min, as previously described [29–31]. Each mouse was judged to be immobile when it ceased struggling, and remained floating motionless in the water, making only those movements necessary to keep its head above water. The average percentage immobility was calculated by a blinded experimenter.
Erythropoietin and NADPH oxidase expression
qRT-PCR analysis of EPO, EPO receptor, and p47phox was performed using ABI PRISM 7500 System (Applied Biosystems, Foster City, CA). PCR Primers and Taqman probes for EPO and p47phox were purchased from ABI (Applied Biosystems). Each reaction (25μl) contained 2.5 μl reaction buffer (10x), 6 mM MgCl2, 0.2 μM dNTP, 0.6 μM each primer, 0.25 μl SureStar Taq DNA Polymerase and 2 μl cDNA dilutions. The cycling condition consisted of 1 cycle at 95°C for 10 min and 40 three-segment cycles (95°C for 30 s, 55°C for 60 s and 72°C for 30 s). Standard curves for gene of interest and housekeeping gene (β-actin) were included in each reaction. We found that the mRNA expression of β-actin was stable after IH or SH exposures. Expression values were obtained from the cycle number (Ct value) using the MX4000 software (Stratagene, La Jolla, CA). EPO, P47phox, and β-actin mRNA were performed in triplicates to determine the Ct-diff. These Ct values were averaged and the difference between the β-actin Ct (Avg) and the gene of interest Ct (Avg) was calculated (Ct-diff). The relative expression EPO and p47phox was analyzed using the 2-ΔΔCT method. Quantitative results were expressed as the mean ± standard deviation (SD).
Animals were deeply anesthetized and perfused intracardially with 4% phosphate-buffered paraformaldehyde. Serial sections were cut on a microtome. The free floating sections were incubated with a goat anti-mouse polyclonal EPO antibody(1:200 dilution; LS Biosciences;LS-C128821) and anti-NeuN (1:1000 dilution; Millipore, clone A60). Immunostained sections were further visualized with FITC-conjugated or rhodamine-conjugated 2nd antibody. Sections were initially assessed using a Nikon Ellipse E800 microscope, and subsequently with a confocal microscope (Leica TCS SP5). To present the expression patterns in a complete fashion, a montage of photomicrographs was assembled using Adobe Photoshop 8.0.
Lipid peroxidation assay
MDA-586 kits (OxisResearch, Portland OR) were used to measure the relative malondialdehyde (MDA) production, a commonly used indicator of lipid peroxidation , in frontal brain cortex according to the manufacturer's instructions. Briefly, after anesthesia with pentobarbital (50 mg/kg intraperitoneally), mice were perfused with 0.9% saline buffer for 5 minutes and the cortex was dissected, snap frozen in liquid nitrogen, and stored at −80°C until assay the following day. Cortical and hippocampal tissues were homogenized in 20 mM phosphate buffer (pH 7.4) containing 0.5 mM butylated hydroxytoluene to prevent sample oxidation. After protein concentration measurements, equal amounts of proteins (2.0–2.5 mg protein from each sample) were used in triplicate to react with chromogenic reagents at 45°C in 500 μL buffer for 2 hours. The samples were then centrifuged and clear supernatants measured at 586 nm. The level of MDA production was then calculated with the standard curve obtained from the kit according to the manufacturer's instructions.
8-hydroxydeoxyguanosine (8-OHDG) tissue levels
Levels of 8-OHDG were measured in frontal brain cortex and hippocampus using a commercially available assay (Cell Biolabs, San Diego, CA). 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 an horseradish peroxidase-conjugated secondary antibody. The 8-OHDG content in the cortical samples was then determined by comparison with the 8-OHDG standard curve.
Erythropoietin tissue levels
Tissue concentrations of EPO were measured in duplicate in hippocampal lysates using a commercially available ELISA assay (Quantikine cat #MEP00, R&D Systems, Inc, Minneapolis, MN). This assay was linear between 22–3,000 pg/ml using a standard calibration curve, and the intra- an inter-individual coefficients of variability were 4.6% and 8.4%, respectively.
Primary neuronal cell cultures
Cortical neuronal cells were prepared from fetal mouse brain cortex at embryonic stage 14.5 days (E14.5). Manually dissociated brain cortical cells were plated in a Petri dish coated with poly-L-ornithine (0.015g/L) in a culture medium including neurobasal medium (Gibco), Mix B27(Gibco), L-glutamine 250uM, glutaMax 250uM, Antibiotic/Amycotic (Gibco) 1%. Half of the media in the wells were removed and replaced with fresh culture medium every 3 days. After 12 days in culture, primary neuronal cells were used for in vitro IH experiments. Cultured cortical neuronal cells were exposed to normoxia or IH respectively in a computer controlled cell incubator chamber that tightly controls O2 concentrations in the cell culture medium and in the cell culture chambers (Reming Bioinstruments, Redfield, NY). For normoxia treatment, cells were cultured under normal cell culture conditions (37°C, 95% air and 5% CO2 in a humid incubator). For IH or SH treatments, cell were treated with either alternations of 35min-5%O2/5%CO2 balance N2 followed by 25min-21% O2/5%CO2 balance N2 (IH) or exposed to 5%O2/5%CO2 balance N2 (SH) for 72 hrs in the presence or absence of pre-treatment with EPO in culture medium (1,500 pg/ml). Cells were collected, and RNA was isolated and subjected to qRT-PCR analysis for NADPH oxidase p47phox subunit expression, as delineated above.
To elucidate the nature of interactions between IH, SH, and RA conditions, all data were initially analyzed by one way ANOVA. First, overall statistical significance was determined for the entire training period between the treatment groups. In addition, either two-way repeated measures ANOVA or MANOVA were used to analyze each trial block, followed by post-hoc Tukey tests. Similar statistical approaches were used to compare probe trial, reference memory, EPM and FST. For all comparisons, a p value <0.05 was considered to achieve statistical significance.
In all the experimental conditions, the data were divided into 6 blocks (containing 3 trials/day). We used a multivariate MANOVA model (SPSS software 17; Chicago) that included latency, pathlength and swim speed and two between factors: (1) Groups (four levels): RA-C, IH-C, RA-EPO, and IH-EPO (2) Condition (two levels): RA or IH. All F statistics are reported using Pillai’s Trace. The interaction of three different factors, i.e., time, condition and group were determined using this mixed model repeated measures MANOVA.